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Pelotas, 2008
Éverton Fagonde da Silva
Avaliação de antígenos recombinantes para o
desenvolvimento de uma vacina contra leptospirose
UNIVERSIDADE FEDERAL DE PELOTAS
Programa de Pós-Graduação em Biotecnologia
Agrícola
Tese
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ÉVERTON FAGONDE DA SILVA
Avaliação de antígenos recombinantes para o
desenvolvimento de uma vacina contra leptospirose
Orientador: Odir Antônio Dellagostin
Co-Orientador: Albert Icksang Ko
Pelotas, 2008
Tese apresentada ao Programa de Pós-
Graduação em Biotecnologia Agrícola da
Universidade Federal de Pelotas, como
requisito parcial à obtenção do título de
Doutor em Ciências (área de conhecimento:
Biologia Molecular).
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Dados de catalogação na fonte:
Ubirajara Buddin Cruz – CRB-10/901
Biblioteca de Ciência & Tecnologia - UFPel
S586a Silva, Éverton Fagonde da
Avaliação de antígenos recombinantes para o
desenvolvimento de uma vacina contra leptospirose /
Everton
Fagonde da Silva
; orientador Odir Antônio Dellagostin ; co-
orientador Albert Icksang Ko. – Pelotas, 2008. – 99f. : il. – Tese
(Doutorado). Programa de Pós-Graduação em Biotecnologia
Agrícola. Centro de Biotecnologia. Universidade Federal de Pelotas.
Pelotas, 2008.
1.Biotecnologia. 2.Vacinas recombinantes. 3.Leptospirose.
4.Proteínas de membrana externa. I.Dellagostin, Odir Antônio. II.Ko,
Albert Icksang. III.Título.
CDD: 614.56
Banca examinadora:
Prof. Dr. Odir Antônio Dellagostin (Orientador), Universidade Federal de Pelotas
Prof. Dr. José Antonio Guimarães Aleixo, Universidade Federal de Pelotas
Prof. Dr. Fábio Pereira Leivas Leite, Universidade Federal de Pelotas
Prof. Dr. Itabajara da Silva Vaz Jr., Universidade Federal do Rio Grande do Sul
AGRADECIMENTOS
Agradeço ao meu Deus e a toda a minha família, pelo apoio e compreensão,
e principalmente pelo amor e carinho durante todos os períodos. Agradeço muito a
Flávia Aleixo Vasconcellos pelo carinho, amizade e apoio durante a fase final do
presente trabalho.
À Universidade Federal de Pelotas, em especial ao Centro de Biotecnologia, e
à Fundação Oswaldo Cruz, em especial ao Centro de Pesquisas Gonçalo Moniz e
Bio-Manguinhos, pela oportunidade de realizar o curso de doutorado e parte da fase
experimental do presente trabalho, respectivamente.
Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq),
Fundação Oswaldo Cruz e ao NIH Fogarty International Center - Global Infectious
Disease Training Program pela concessão da bolsa de estudos.
Ao professor Odir Antônio Dellagostin pela orientação e dedicação durante o
período de execução deste trabalho. Além de um exemplo de profissional, tu és e
sempre serás um grande amigo meu, principalmente por estares presente nos
momentos mais difíceis do doutorado, ‘’dando o maior apoio’’. Além disso, estivesse
presente também nos momentos de felicidade, compartilhando os nossos ótimos
resultados, que eu espero que se repitam por muitos e muitos anos durante a nossa
convivência profissional.
Ao professor Albert I. Ko pela oportunidade de realizar grande parte da fase
experimental deste trabalho, confiando na minha capacidade profissional e na minha
dedicação, não poupando esforços para a minha orientação. Muito obrigado pelo
apoio e ajuda durante toda a nossa convivência em Salvador.
Aos demais professores do Centro de Biotecnologia, pelos ensinamentos e
pela convivência, em especial aos professores José Antonio Guimarães Aleixo e
Carlos Gil Turnes. Agradeço também a Alegani, Ivete e Michele pela convivência e
por todo o apoio necessário.
Aos professores, pesquisadores, alunos de pós-graduação e iniciação
científica da Fiocruz/BA, e também aos pesquisadores de Bio-Manguinhos que
contribuíram para a minha formação profissional. Agradeço em especial ao professor
Mitermayer Reis, que abriu as portas do LPBM para a execução do meu trabalho,
sendo um exemplo durante o período de nossa convivência. Em especial também, a
três profissionais e amigos que conquistei durante o mesmo período, ao Cleiton
Santos, Daniel Athanazio e ao Adriano Queiroz, muito obrigado por tudo. Obrigado
também ao Alan John e a Flávia McBride pelo convívio e por toda a ajuda.
Aos colegas da pós-graduação e do laboratório de Biologia Molecular, em
especial para a Fabiana, Gustavo, Daiane, Luciano, Sibele, Simone, Vanusa,
Silvana e Samuel Félix, e aos alunos da iniciação científica Michel, Robson, Samuel
Ribeiro, Danieli, Tessália e Vanessa. Agradeço também aos colegas e amigos dos
demais laboratórios pela saudável convivência durante este período.
Não posso deixar de agradecer, também, a todos aqueles que direta ou
indiretamente contribuíram para a realização deste trabalho.
Muito Obrigado a todos!
RESUMO
SILVA, Éverton Fagonde da. Avaliação de antígenos recombinantes para o
desenvolvimento de uma vacina contra leptospirose. 2008. 99 f. Tese
(Doutorado) - Programa de Pós-Graduação em Biotecnologia Agrícola. Universidade
Federal de Pelotas, Pelotas.
A leptospirose é uma doença infecciosa aguda causada por espiroquetas
patogênicas do gênero
Leptospira
. Esta zoonose ocorre em todo o mundo, sendo
mais freqüente em regiões tropicais e subtropicais onde a temperatura elevada e as
chuvas favorecem a sua transmissão aos hospedeiros suscetíveis. No Brasil, a
leptospirose ocorre com maior freqüência em áreas urbanas e regiões
metropolitanas. O alto custo hospitalar, os dias de trabalho perdidos e o elevado
grau de letalidade demonstram a importância desta enfermidade para a saúde
pública. A vacinação de animais é amplamente realizada, porém, até o momento,
não existe uma vacina para uso humano contra leptospirose no Brasil. O objetivo
deste trabalho foi avaliar antígenos recombinantes de
L. interrogans
, produzidos em
Escherichia coli,
em modelo animal de leptospirose, visando o desenvolvimento de uma
nova vacina. Para isso, isolados patogênicos de diferentes espécies de
Leptospira
foram caracterizados quanto à virulência e a capacidade de reproduzir a doença em
hamsters. Posteriormente sete proteínas recombinantes foram produzidas e
avaliadas quanto à antigenicidade, imunogenicidade e imunoproteção. Todas as
proteínas testadas mostraram-se antigênicas e imunogênicas, porém apenas duas
conferiram proteção contra desafio com uma dose letal de
L. interrogans
sorovar
Copenhageni: um fragmento da proteína LigA (LigANI) e uma lipoproteína referida
como Lip1. LigANI conferiu imunoproteção de 67 a 100% (P<0,05) quando
administrada com adjuvante de Freund, porém não impediu a colonização renal pela
bactéria. A lipoproteína Lip1, administrada com hidróxido de alumínio como
adjuvante, conferiu imunoproteção de 100% contra mortalidade em hamsters
(P<0,001), e também foi capaz de induzir imunidade esterilizante. Estes resultados
indicam que LigANI e Lip1 são candidatos potenciais para uma vacina recombinante
contra leptospirose humana e animal.
Palavras-chave: Leptospirose. Vacina recombinante. Immunoproteção.
ABSTRACT
SILVA, Éverton Fagonde da. Evaluation of recombinant antigens for the
development of a vaccine against leptospirosis. 2008. 99 f. Tese (Doutorado) -
Programa de Pós - Graduação em Biotecnologia Agrícola. Universidade Federal de
Pelotas, Pelotas.
Leptospirosis is an acute infectious disease caused by pathogenic spirochetes of the
genus
Leptospira
. This zoonosis occurs worldwide, however it is most frequently found
in tropical and subtropical regions where the temperature and rainfall facilitate
transmission of the bacteria to susceptible hosts. In Brazil, leptospirosis occurs
mainly in urban areas and metropolitan regions. Hospitalization cost, loss of work
days and the high lethality rate demonstrate the importance of this disease for public
health. Vaccination of animals is widely used, however, to date there is no vaccine
for human leptospirosis in Brazil. The objective of this work was to evaluate
recombinant
L. interrogans
antigens produced in
Escherichia coli
, in an animal model of
leptospirosis, aiming at the development of a new vaccine. For that, pathogenic
isolates of different
Leptospira
species were characterized regarding virulence and
capacity for reproducing the disease in hamsters. Then, recombinant proteins were
produced and evaluated regarding antigenicity, immunogenicity and
immunoprotection. All the proteins tested were antigenic and immunogenic, however
only two were able to protect hamsters against challenge with a lethal dose of
L.
interrogans
sorovar Copenhageni: a fragment of the LigA protein (LigANI) and a
lipoprotein referred to as Lip1. LigANI conferred protection of 67 to 100% (P<0.05)
when administered with Freund’s adjuvant, however it did not avoid renal
colonization by the bacterium. The lipoprotein Lip1, administered with aluminum
hydroxide as adjuvant, conferred immunoprotection of 100% against mortality in
hamsters (P<0.001) and it was able to induce a sterilizing immunity. These findings
indicate that LigANI and LipL1 are potential vaccine candidates against human and
veterinary leptospirosis.
Keywords: Leptospirosis. Recombinant vaccine. Immunoprotection.
SUMÁRIO
AVALIAÇÃO DE ANTÍGENOS RECOMBINANTES PARA O
DESENVOLVIMENTO DE UMA VACINA CONTRA A LEPTOSPIROSE ..........
1
RESUMO ..............................................................................................................
4
ABSTRACT .........................................................................................................
5
1 INTRODUÇÃO GERAL ....................................................................................
8
2 ARTIGO 1 .........................................................................................................
1
4
CHARACTERIZATION OF VIRULENCE OF
Leptospira
ISOLATES IN
HAMSTER MODEL …………………………………………………………………...
14
ABSTRACT ......................................................................................................
1
6
INTRODUCTION ..............................................................................................
1
7
MATERIALS AND METHODS ..........................................................................
1
8
RESULTS .........................................................................................................
19
DISCUSSION ...................................................................................................
2
0
ACKNOWLEDGMENTS ...................................................................................
2
2
REFERENCES .................................................................................................
2
3
3 ARTIGO 2 .........................................................................................................
3
4
THE TERMINAL PORTION OF LEPTOSPIRAL IMMUNOGLOBULIN-LIKE
PROTEIN LigA CONFERS PROTECTIVE IMMUNITY AGAINST LETHAL
INFECTION IN THE HAMSTER MODEL OF LEPTOSPIROSIS………………...
34
ABSTRACT ......................................................................................................
3
6
INTRODUCTION ..............................................................................................
3
7
MATERIALS AND METHODS ..........................................................................
39
RESULTS .........................................................................................................
4
4
DISCUSSION ...................................................................................................
4
7
ACKNOWLEDGMENTS ...................................................................................
5
0
REFERENCES .................................................................................................
5
1
4 ARTIGO 3 .........................................................................................................
66
VACCINATION WITH A NOVEL LIPOPROTEIN CONFERS PROTECTIVE
IMMUNITY AGAINST LETHAL INFECTION IN THE HAMSTER
MODEL OF LEPTOSPIROSIS………………………………………..……………...
66
ABSTRACT ......................................................................................................
67
REFERENCES .................................................................................................
72
5 CONCLUSÕES ................................................................................................
79
6 REFERÊNCIAS ................................................................................................
8
0
7 ANEXO .............................................................................................................
89
8
1. INTRODUÇÃO GERAL
A leptospirose é uma zoonose, com importância mundial, causada por
leptospiras patogênicas (BHARTI et al., 2003; WHO, 2003). Apesar de sua ampla
distribuição geográfica, é na América Latina, na África e na Ásia, que a sua
ocorrência torna-se mais preocupante. Este comportamento endêmico da
enfermidade nestas regiões do mundo está relacionado principalmente com fatores
climáticos e ambientais, além da existência de fatores sociais, econômicos e
culturais que contribuem para o estabelecimento e a disseminação da leptospirose
(SEHGAL et al., 1995; FAINE et al., 1999; LEVETT, 2001; VINETZ, 2001; McBRIDE
et al., 2005).
Na última década, o aparecimento de casos humanos em países como
Nicarágua (CDC, 1995; TREVEJO et al., 1998), Índia e sudeste da Ásia (WHO,
2000), Estados Unidos (CDC, 1998) e na Malásia (CDC, 2001), fez ressurgir o
interesse internacional na leptospirose. Atualmente, a leptospirose é considerada
como uma das doenças infecciosas emergentes no mundo (WHO, 1999; LEVETT,
2001; MEITES et al., 2004), embora alguns autores questionem se a leptospirose é
uma enfermidade re-emergente ou se está sendo re-descoberta (LEVETT, 1999;
MEITES et al., 2004).
Leptospiras são tradicionalmente classificadas de acordo com sua
semelhança antigênica (DIKKEN; KMETY, 1978; TERPSTRA, 1992; KMETY;
DIKKEN, 1993) e até o momento, houve a descrição de mais de duzentos sorovares
patogênicos que podem causar a enfermidade em diferentes hospedeiros (MOREY
et al., 2006). Mais recentemente, a classificação molecular tem sido amplamente
considerada, na qual o gênero foi dividido em espécies, com base em peculiaridades
do DNA das diferentes cepas isoladas no mundo (YASUDA et al., 1987;
RAMADASS et al., 1992; PEROLAT et al., 1998; BRENNER et al., 1999; LEVETT et
al., 2006). Embora a reclassificação das leptospiras, usando os achados genéticos,
possa fornecer uma informação taxonômica importante, a classificação sorológica é
a mais familiar para epidemiologistas e clínicos tanto na área humana quanto animal
(BHARTI et al., 2003).
Vacinas contra a leptospirose estão disponíveis para o uso humano e
veterinário (FAINE et al., 1999) desde o século passado. Imunizações com
bacterinas têm induzido proteção contra infecção com
Leptospira
, evidenciando uma
9
resposta imune humoral protetora, sendo essa, uma proteção sorovar-específica
(MIDWINTER et al., 1990). Alguns países como Cuba (MARTÍNEZ et al., 2004;
GONZÁLES et al., 2005) e China (LEVETT, 2001) desenvolveram vacinas baseadas
em preparações com bacterinas para o uso humano, utilizando exemplares dos
sorogrupos mais importantes epidemiologicamente em seus países. Como estas
preparações não foram testadas em ensaios clínicos controlados, geraram
discordâncias sobre a verdadeira eficácia dessas vacinas, e além disso, o custo de
produção em larga escala e a falta de homogeneidade dessas preparações são
considerados uma significativa limitação dessas vacinas (McBRIDE et al., 2005).
Na área veterinária, o uso de bacterinas está difundido em todos os
continentes (ELLIS et al., 1989; BOLIN; ALT, 2001). Preparações mono e
polivalentes encontram-se disponíveis comercialmente, sendo produzidas por
diferentes fontes laboratoriais. Estas vacinas apresentam as mesmas limitações das
preparações para humanos, evidenciando um baixo espectro de proteção e
necessitando de revacinação dos animais a cada 6 meses ou 1 ano (FAINE et al.,
1999; NAIMAN et al., 2002; ANDRÉ-FONTAINE et al., 2006).
Uma importante limitação deste tipo de estratégia empregada para humanos
e animais é a utilização de cepas patogênicas com múltiplas passagens in vitro nas
preparações, as quais sofrem modificações em sua superfície e a perda da sua
virulência em modelo animal (HAAKE et al., 1991). Como importante conseqüência,
essas vacinas carecem de alvos importantes que são expressos somente durante
uma infecção por cepas virulentas (BARNETT et al., 1999; GUERREIRO et al.,
2001).
Devido a necessidade da implementação de técnicas modernas de
diagnóstico e de medidas preventivas mais eficazes para o controle desta importante
enfermidade, países como China e Brasil investiram no seqüenciamento do genoma
de duas cepas pertencentes à espécie
L. interrogans
e ao sorogrupo
Icterohaemorrhagiae, as cepas Lai 56601 (REN et al., 2003) e Fiocruz L1-130
(NASCIMENTO et al., 2004), consideradas como agentes endêmicos e responsáveis
pela grande maioria dos casos graves da leptospirose humana em seus países. O
seqüenciamento do genoma da cepa Fiocruz L1-130 foi realizado em uma
colaboração que envolveu a Fundação Oswaldo Cruz (RJ e BA), Instituto Butantan e
a Universidade Federal de Pelotas, entre outras instituições nacionais e
internacionais. Mais recentemente, o genoma de duas cepas do sorovar Hardjo
10
pertencentes à espécie
L. borgpetersenii
, importante causa da leptospirose bovina no
mundo, também tiveram o seu genoma seqüenciado e comparado com os outros
genomas disponíveis (BULACH et al., 2006). O genoma de cada uma das quatro
leptospiras seqüenciadas até o momento consiste em dois cromossomos circulares,
sendo de maior tamanho quando comparado ao de outras espiroquetas como
Treponema spp
(FRASER et al., 1998) e
Borrelia spp
(FRASER et al., 1997).
Estudos sobre a superfície das leptospiras revelaram que elas possuem duas
membranas, uma membrana externa (OM) e uma citoplasmática ou membrana
interna (IM). Semelhante ao que ocorre com as bactérias gram-positivas, o
Peptoglicano (PG) da parede celular está associado com a IM, porém, a superfície
das leptospiras está coberta por Lipopolisacarídeos (LPS), similarmente ao que
ocorre com as bactérias gram-negativas, mas com uma toxicidade dez vezes menor
(LEVETT, 2001).
A OM das leptospiras contém muitas lipoproteínas já caracterizadas como
importantes alvos para o desenvolvimento de vacinas, como LipL32/HAP-1 (HAAKE
et al., 2000; BRANGER et al., 2001; SEIXAS et al., 2007a), LipL21 (CULLEN et al.,
2003), LipL41 (SHANG et al., 1996) e a porina OmpL1 (HAAKE et al., 1993; SHANG
et al., 1995). Além destes, o seqüenciamento do genoma de
L. interrogans
Fiocruz L1-
130, revelou genes que codificam para outros tipos de proteínas de membrana
externa (OMPs), incluindo as OMPs TonB-dependentes, fator de membrana externa
(OMF), proteína de fusão de membrana (MFP) e o transportador da membrana
interna (CzcA), além de identificar novos alvos potenciais de OM, que podem ser
candidatos para uma vacina contra a leptospirose (NASCIMENTO et al., 2004;
GAMBERINI et al., 2005).
Anteriormente ao seqüenciamento do genoma da cepa Fiocruz L1-130 e das
demais, alguns possíveis fatores de virulência desta cepa já haviam sido descritos
(GUERREIRO et al., 2001; MATSUNAGA et al., 2003). A resposta imune humoral de
humanos em decorrência da infecção por leptospiras revelou proteínas que são
expressas durante a infecção. Mediante a construção de bibliotecas de expressão
com genes de Fiocruz L1-130 e
L. kirschneri
Grippotyphosa RM52 (HAAKE et al.,
1999), utilizando soros de pacientes humanos em fase convalescente da
enfermidade, foram identificadas além de proteínas de choque térmico e
chaperonas, três novos genes que codificavam para as já descritas “Bacterial
immunoglobulin-like proteins (Big)” (MATSUNAGA et al., 2003). Estes genes
11
encontrados nas leptospiras foram denominados de “Leptospiral immunoglobulin-like
(Lig)”, e codificam para as proteínas LigA, LigB e LigC, encontradas somente em
cepas patogênicas (PALANIAPPAN et al., 2002; MATSUNAGA et al., 2003). Estudos
de microscopia imunoeletrônica confirmaram que as proteínas Lig estão localizadas
na superfície da leptospiras (MATSUNAGA et al., 2003). Outra descoberta
importante, é que todas as três proteínas Lig possuem domínios encontrados nas
proteínas Intimina de
E. coli
enterotoxigênica (FRANKEL et al., 1996; LUO et al.,
2000), Invasina de
Yersinia pseudotuberculosis
(ISBERG et al., 1987; HAMBURGER et
al., 1999) e BipA de
Bordetella sp
. (STOCKBAUER et al., 2001), que são fatores de
virulência nestes microrganismos.
Recentemente, foi demonstrado que as proteínas Lig são adesinas (CHOY et
al., 2007) e que a expressão delas é fortemente regulada pela osmolaridade
(MATSUNAGA et al., 2005). Por outro lado, o seqüenciamento dos genes lig de
cepas virulentas isoladas de humanos e animais, pertencentes a 4 espécies
patogênicas (
L. interrogans, L. noguchii, L. weilli e L. borgpetersenii
), e a comparação com as
seqüências disponíveis em bancos de dados (GenBank) para as demais espécies,
revelaram a presença do gene ligB em todas as espécies analisadas, do gene ligA
nas espécies
L. interrogans e L. kirshneri
e o gene ligC somente em
L. interrogans, L.
kirshneri, L. weilli
e em alguns sorovares da espécie
L. noguchii
(CERQUEIRA, 2006).
Novas estratégias, baseadas em antígenos recombinantes, surgem como
uma alternativa para o controle de enfermidades bacterianas. Como por exemplo,
imunizações com determinantes protéicos associados com a membrana externa,
têm mostrado respostas protetoras em modelos animais experimentais, contra
infecções como a Doença de Lyme (SIGAL, 1999; THANASSI; SCHOEN, 2000) e
Doença meningocócica B (PIZZA et al., 2000). Para a leptospirose, algumas
preparações já foram testadas em modelo animal, seja como vacina de subunidade
(HAAKE et al., 1999; KOIZUMI; WATANABE, 2003; PALANIAPPAN et al., 2006),
como vacina de DNA (BRANGER et al., 2001; BRANGER et al., 2005; FAISAL et al.,
2008) ou como vacina vetorizada por BCG recombinante (SEIXAS et al., 2007b),
mas nenhuma destas preparações conferiu proteção de amplo espectro até o
presente momento.
Neste contexto, visto que importantes determinantes de virulência são
expressos apenas durante a infecção no hospedeiro, a hipótese deste trabalho foi de
que as proteínas Lig e os demais alvos protéicos de superfície selecionados a partir
12
do projeto genoma de
L. interrogans
sorovar Copenhageni, são potenciais candidatos a
uma vacina recombinante contra a leptospirose.
Para isso, o objetivo geral deste trabalho foi o de avaliar antígenos
recombinantes de
L. interrogans
para o uso em uma vacina. Como objetivos
específicos, destacam-se: (1) Padronizar e validar o uso de isolados locais em
experimentos de desafio em hamsters; (2) Avaliar a imunogenicidade das proteínas
Lig e a sua capacidade em induzir proteção em modelo animal suscetível e (3)
Avaliar lipoproteínas, produzidas de forma recombinantes em
E. coli
, quanto à
capacidade de induzir proteção em modelo animal suscetível.
Inicialmente, apresentamos o artigo 1 que foi submetido ao periódico
Vaccine, onde testou-se a virulência de diferentes isolados de
Leptospira
, obtidos de
humanos, caninos e camundongo do Brasil (Salvador/BA e Pelotas/RS), e que são
representantes de importantes sorogrupos, tanto para saúde pública como para a
área veterinária. A padronização da dose letal 50% para cada um dos isolados
virulentos em hamsters, objetivou garantir a reprodutibilidade dos resultados em
futuros experimentos que utilizam esse modelo animal para o teste de candidatos a
uma vacina de amplo espectro contra a leptospirose.
Em seguida, o artigo 2 trata da avaliação da imunogenicidade de fragmentos
das proteínas LigA e LigB, bem como a sua capacidade em induzir uma resposta
imune protetora contra leptospirose em hamsters, quando utilizados como vacina de
subunidade. Até o momento, esse é o primeiro trabalho que apresenta uma
evidência conclusiva de que a imunização com proteínas recombinantes purificadas
confere proteção em modelo animal suscetível (hamsters) à leptospirose letal. Esse
trabalho, assim como o anterior, foi realizado em Salvador, na Fiocruz/BA, nos dois
primeiros anos do doutorado, e fez parte de um projeto maior denominado de
‘’Desenvolvimento de uma vacina contra a leptospirose’’, onde o Cenbiot/UFPel é
colaborador de outras instituições nacionais como a FIOCRUZ (RJ, BA, Bio-
Manguinhos), e internacionais como a Universidade da Califórnia (UCLA/USA) e a
Universidade de Cornell (New York/USA). Este artigo foi publicado no periódico
Vaccine.
No artigo 3, utilizando-se uma estratégia semelhante a do artigo 2, quatro
alvos caracterizados como candidatos potenciais a uma vacina contra a leptospirose
foram avaliados como vacina de subunidade no mesmo modelo animal. Os
resultados mostraram que Lip1 recombinante foi capaz de proteger os hamsters
13
imunizados contra o desafio com dose letal de leptospiras. Este trabalho foi
realizado integralmente no Cenbiot/UFPel, e será submetido como uma Short
communication ao periódico Clinical and Vaccine Immunology.
14
2. ARTIGO 1
CHARACTERIZATION OF VIRULENCE OF Leptospira ISOLATES IN A
HAMSTER MODEL
(Artigo Submetido ao Periódico Vaccine)
15
CHARACTERIZATION OF VIRULENCE OF Leptospira ISOLATES IN A
HAMSTER MODEL
Éverton F. Silva
a,b,*
; Cleiton S. Santos
b
; Daniel A. Athanazio
b
; Núbia Seyffert
a
;
Fabiana K. Seixas
a
; Gustavo M. Cerqueira
a
; Michel Q. Fagundes
a
; Claudiomar
S. Brod
c
; Mitermayer G. Reis
b
; Odir A. Dellagostin
a
; Albert I. Ko
b,d
a
Centro de Biotecnologia, Universidade Federal de Pelotas, UFPel, Brazil
b
Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Brazil
c
Centro de Controle de Zoonoses, Faculdade de Medicina Veterinária, UFPel, Brazil
d
Division of International Medicine and Infectious Disease, Weill Medical College of
Cornell University, New York, USA.
Address for correspondence: Universidade Federal de Pelotas. Centro de
Biotecnologia. Campus Universitário. Caixa Postal 357, CEP: 96010-900. Pelotas,
RS, Brazil. Phone: +55 53 32757587. Fax: +55 53 32757350. E-mail address:
16
ABSTRACT
Effort has been made to identify protective antigens in order to develop a
recombinant vaccine against leptospirosis. Several attempts failed to conclusively
demonstrate efficacy of vaccine candidates due to the lack of an appropriate model
of lethal leptospirosis. The purposes of our study were: (i) to test the virulence of
leptospiral isolates from Brazil, which are representative of important serogroups that
cause disease in humans and animals; and (ii) to standardize the lethal dose 50%
(LD
50
) for each of the virulent strains using a hamster (Mesocricetus auratus) model,
ensuring reproducibility of this procedure in future assays. Five of seven Brazilian
isolates induced lethality in a hamster model, with inocula lower than 200 leptospires.
Furthermore, we conducted histopathological examination and observed the
reproducibility of typical lesions found in both natural and experimental leptospirosis.
Results described here demonstrated the potential use of Brazilian isolates as highly
virulent strains in challenge experiments using hamster, an appropriate animal model
for leptospirosis. Furthermore these strains may be useful in heterologous challenge
studies which aim to evaluate cross-protective responses induced by subunit vaccine
candidates.
Keywords: leptospirosis; human; dog; mouse; isolation; animal model; challenge.
17
1. INTRODUCTION
Leptospirosis is an infectious disease caused by pathogenic leptospires that
are transmitted directly or indirectly to humans and animals. Leptospirosis occurs
worldwide but it is more commonly associated with tropical and subtropical areas [1].
The disease is found mainly wherever humans come into contact with the urine of
infected animals or an urine-contaminated environment [2]. Exposure to leptospires
may be associated to water sports, sporadic exposure in urban areas of developed
countries, outbreaks associated with rain season and floods in urban areas with poor
sanitary conditions, and it also occours as an endemic disease in rural areas in Latin
America and Southeast Asia [2-4]. The clinical presentation of human infection
ranges from olygosymptomatic or undifferentiated febrile illness to severe forms of
Weil’s, with an overall 10-15% case fatality, and severe pulmonary hemorrhagic
syndrome (SPHS), with ≥50% case fatality [5,6].
Whole genome sequences are now available for Leptospira interrogans
serovars Lai and Copenhageni and L. borgpetersenii serovar Hardjo [7-9]. Both
China [9] and Brazil [8] have made research in leptospirosis a priority in order to
address this emerging public health problem. In the case of Brazil, a national
multiinstitutional initiative led to the whole genome sequencing of L.interrogans
serovar Copenhageni, the agent for epidemics of severe leptospirosis in Brazilian
urban centers [10,11]. A major goal has been the use of genome information to
identify targets for a subunit based vaccine [12]. Ideally an effective vaccine would
induce induce cross-protection against the range of serovars of public health
importance. Encorauging data using recombinant proteins and DNA vaccines in
different animal models has already been published [13-19].
Brazilian human isolates of serovar Copenhageni have been used in recent
studies on pathogenesis and proteomics [20-27]. However, there is a wide diversity
of Leptospira serovars that constitute etiological agents of leptospirosis, specialy in
rural settings. To date, there is little information on virulence of pathogenic isolates
using a standard animal model. This is a critical issue for the evaluation of vaccine
candidates that elicit potential cross immunoprotection response.
In this context, we tested the virulence of Leptospira isolates obtained from
humans, dogs and from a mouse, representative of serogroups important for public
health and veterinary areas; and standardized the LD
50
for each virulent strain in
hamster (Mesocricetus auratus) model, ensuring the reproducibility of this procedure
18
for use in future challenge experiments. Since recent data suggest histopathology is
an important indicator of outcome during sublethal disease [15,28], we also
conducted pathologic examination of the hamsters experimentally infected by the
virulent strains.
2. MATERIALS AND METHODS
2.1. Bacteria
All l
eptospires were cultivated in liquid Ellinghausen-McCullough-Johnson-
Harris (EMJH) medium (Difco Labratories) at 29 °C, and leptospires were counted in
a Petroff-Hauser counting chamber (Fisher Scientific) as previously described [29]. L.
interrogans serovar Copenhageni strain Fiocruz L1-130 [11] and six isolates from
Pelotas city in the South of Brazil, previously serogrouped (Table 1), were passaged
in hamster, and stored at both -70 °C and in liquid nitrogen until use. In order to
minimize variability on virulence due to re-adaption to culture medium, each isolate
received a specific notation as function of the number of in vivo and in vitro
passages. Thus, for standardization and reproducibility of future experiments, Fiocruz
L1-130 was passaged in hamsters four times and then three times in EMJH medium.
Aliquots were passaged four times in liquid medium prior to their use as a low-
passage-number isolate for the infection experiments. For example, the 4.7 code
refers to the number of passages in hamsters (4) and the number of in vitro
passages (7), respectively. Other strains had a distinct number of passages in
hamsters and in vitro (Table 1).
2.2. Animals and virulence test design
Male and female Golden Syrian hamsters (Fiocruz/BA) were used in all
experiments. For virulence evaluation of the clinical isolates, the following outcomes
were measured: onset of clinical symptoms, number of deaths, days until death and
subsequent re-isolation of leptospires. Two four-week old hamsters, weighing
approximately 55 g, were infected intraperitoneally with 10
8
leptospires. All animals
were monitored daily for the presence of clinical signs, including evidence of external
hemorrhage, dehydration, ruffled hair coat, decreased activity and isolation. In these
conditions, hamsters were euthanized and kidney tissue was cultured at 29 °C in
liquid EMJH media without antibiotics. After two or three sub-cultures in liquid media,
each strain was distributed in aliquots of 1 ml and stored at -70 °C and in liquid
19
nitrogen. This project and all animal experiments were approved by the Committee
for Animal Care and Use (CEUA/Fiocruz).
2.3. LD
50
experimental design
For all experiments, eight to nine-week old hamsters, matched by sex for each
dilution, were infected intraperitoneally with 10 fold serial dilution (10
5
to 10
0
).
Animals were monitored daily for clinical outcome until 28 days post-infection. After
this, LD
50
was calculated by the method of Reed and Muench [30]. Negative control
animals were injected with the same volume of sterile EMJH media. All strains were
evaluated in at least three independent experiments.
2.4. Histopatologic analyses
In order to investigate whether inoculation with the strains could reproduce
histopathological changes typical of experimental leptospirosis, we performed
necropsies of the first two animals appearing moribund after infection by each strain
and compared their histopathology with samples from two non infected hamsters of
the same age euthanized nine days after inoculation of 1 ml sterile EMJH media.
Neutral-buffered formalin fixed paraffin-embedded samples were sectioned and
stained with Hematoxilin and Eosin (HE) and silver impregnation by Warthin-Starry
technique [2].
3. RESULTS
Strains Fiocruz L1-130, Kito, Cascata, Hook and Bonito (Table 1) produced
lethal infection in all hamsters tested. Strains Isoton and Skoll were avirulent and did
not cause disease or colonization in hamsters in three challenge experiments. Kito
and Bonito had the lowest LD
50
(<10 leptospires). However, all virulent strains
induced disease and led to death with an inoculum containing less than 200
leptospires. L. interrogans serovar Copenhageni strain Fiocruz L1-130 had a LD
50
of
120 leptospires in females and 30 in males. L. interrogans serogroup Canicola strain
Kito, had a LD
50
of 2.4
in females and 2.7
in males. L. noguchii serogroup Bataviae
strain Cascata had a LD
50
of 56.2
in females and 33.8
in males. L. noguchii
serogroup Australis strain Hook had a LD
50
of 10
in females and 100
in males. L.
noguchii serogroup Autumnalis strain Bonito had a LD
50
of 1.7
in females and 3.1
in
males (Table 2).
20
Clinical signs were observed from the 4
th
day post infection (d.p.i.) and the
mean period of death of hamsters ranged from 7 to 22 d.p.i. However, animals
experimentally infected with strains belonging to L. interrogans died between 7 and
14 d.p.i. while strains belonging to L. noguchii caused death in hamsters 7 to 22 d.p.i
(Table 3). A survival curve from representative experiments is shown in figure 1.
Hamsters infected with virulent strains developed acute lethal infection
characterized by hepatic and renal complications. Acute damage of tubular epithelia
with cell swelling in proximal segments was observed as a common feature in
moribund hamsters before 10 d.p.i (Fig. 2A). In contrast, hamsters which died after
10 d.p.i. exhibited multifocal regenerative changes in tubular epithelia of renal cortex
and moderate nephritis with infiltrates of leukocytes and histiocytes most often
distributed around small arteries. All moribund animals exhibited marked dissociation
of hepatic trabecula and had hepatocytes which undergone reactive changes such as
cytoplasmic size variation, prominent nucleoli and binucleation (Fig. 2B).
Macroscopic pulmonary and widespread bleeding was found in animals which
received inocula of 10
3
or higher. This was observed in all strains. Although gross
hemorrhages were not detected during necropsy procedure of hamsters which
received inocula of less than 10
3
, microscopic foci of alveolar hemorrhage were
observed in all animals regardless of the inoculated strain. None of these features
were seen in health control animals (Fig. 2C).
4. DISCUSSION
Increasing interest on the development of a vaccine against leptospirosis
emerged in last few years. The field was further stimulated by the whole genome
sequencing of four strains from two pathogenic Leptospira species. Promising results
have been reported by the use of recombinant proteins in hamster [13-15,19]. In
addition, naked DNA and adenovirus used as a vaccine vector for a leptospiral
antigen have also been evaluated in gerbil model [18]. In these studies, the ability of
sub-unit vaccine candidates to induce immunoprotection against heterologous
serovars was not evaluated. However, a recent study reported heterologous
protection in the gerbil model using plasmids encoding LipL32 (HAP1) from serovars
Autumnalis and Grippotyphosa. Immunization with this construct conferred protection
against L. interrogans serovar Canicola challenge [16]. A more complete evaluation
of the capacity of vaccine candidates to induce cross-protective response requires a
21
well-characterized panel of virulent strains representing serovars of public health and
veterinary importance. We report here a suitable model for evaluation of vaccine
candidates against a wider range of pathogenic serovars.
In our study, we characterized five highly virulent Leptospira strains Hamster
challenge protocols used in vaccine evaluations in mice and hamster models have
often required inoculi as high as 10
6
-10
8
leptospires to induce death [15,17], and
when used, do not induce mortality in all control animals. For example, challenge of
an 10
8
inoculum of a serovar Pomona strain induced death in 25-57% of
unvaccinated hamsters (43-75%) [15,28,31], which in turn complicate the
conclusions on the statistical significance of the effectivenes of the candidate
vaccines evaluated in these assays. The low virulence of experimental challenge
model observed in prior studies may be due to the non-susceptibility of animal model
(mouse) [17] or the low virulence of strain used. Protective effects would be better
evaluated using virulent strains in a suitable experimental model which uses a
susceptible host, such as the hamster. In a previous evaluation of homologous
protection using Fiocruz L1-130 strain, fragments of leptospiral immunoglobulin-like
proteins prevented lethal disease in hamsters while unvaccinated controls showed a
high rate of lethality (100% of 76 animals infected with 10
3
challenge) [14].
L. interrogans Copenhageni Fiocruz L1-130 strain was isolated from a patient
identified during the flood-associated outbreak of leptospirosis in Salvador, Brazil, in
1996 [11]. This isolate was also the target of a multicenter initiative to sequence its
genome [8]. Additionally, it has been used for studies on pathogenesis and
leptospiral proteomics [20-21,26-27,32-34]. Serovar Copenhageni is the most
common leptospiral isolate in Salvador and also in other urban areas of Brazil [10-
11,35-36] and its low LD
50
demonstrates that it is a highly virulent pathogen for
hamsters, which has been used in previous immunoprotection experiments [14].
L. interrogans serovar Canicola is the second most important agent for urban
leptospirosis in Brazil [10]. In our study, we could demonstrate it as a highly virulent
strain for hamsters with a general LD
50
of 2.5 leptospires. Serovar Canicola may
cause severe disease in animals and has already been associated to a pulmonary
hemorrhage human leptospirosis outbreak in rural areas of Nicaragua. It has been
used in a recent study employing hamster model to evaluate the protection conferred
by serovar canicola genes presented as DNA vaccines [37].
22
L. noguchii is a pathogenic species present in the American continent and has
been recently reported in the South of Brazil [38]. It had been previously isolated
from human, armadillo, toad, spiny rat, opossum, nutria, Mustela nivalis, cattle and
sheep, showing a wide variety of domestic and wild hosts [2,38,39]. Strains Bonito
and Cascata were isolated from human patients with clinical leptospirosis, while
Hook strain was isolated from a stray dog with lethal leptospirosis. All strains were
virulent for hamsters. Conversely, L. noguchii strain Caco, a previous reported isolate
from sheep, was not virulent in this animal model [38].
There is no clear explanation for the lack of virulence of strain Isoton, an
isolate from the blood culture of a patient with severe leptospirosis. Since avirulent
leptospires may colonize renal tissues of mammalian hosts [40,41], it was not an
unexpected finding the lack of virulence in the strains Skoll from an asymptomatic
mouse. In any case, the lack of virulence of these strains precludes their use in
immunoprotection experiments.
Histopathological analyses found that all virulent strains were able to
reproduce the classical features of the disease, which was already described in
human and experimental leptospirosis [43-47]. The presence of microscopic
pulmonary hemorrhage in the absence of gross features has also been reported in
humans and in guinea pigs [25,44]. The pattern of acute cell swelling in fulminant
disease and a picture of multifocal regeneration tubular foci and interstitial nephritis in
more prolonged illness have also been reported in humans [43].
In conclusion, we characterized the virulence of five clinical isolates of
Leptospira belonging to five different serogroups. These highly virulent strains are
currently been used in experiments aiming at evaluating homologous and
heterologous protection induced by killed whole-cell and recombinant vaccine
candidates against acute lethal leptospirosis in the hamster model.
ACKNOWLEDGEMENTS
This work was supported by CAPES Foundation (Brazilian Government), Bio-
Manguinhos and the Oswaldo Cruz Foundation (09224-7 and PDTIS RVR05), the
Brazilian National Research Council (01.06.0298.00 3773/2005, 420067/2005),
Research Support Foundation for the State of Bahia, and the National Institutes of
Health (5 R01
AI052473, 2 D43 TW-00919).
23
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28
TABLE 1 – Strains used in experiments.
Species Serogroup Strain Source
Sample
isolated
Number of
passages*
L. interrogans Icterohaemorrhagiae L1-130 Human
Blood 4.7
L. interrogans Djasiman Isoton Human
Blood -
L. interrogans Australis Skoll Mouse Kidney -
L. interrogans Canicola Kito Dog Urine 2.5
L. noguchii Australis Hook Dog Kidney 4.5
L. noguchii Bataviae Cascata Human
Blood 2.5
L. noguchii Autumnalis Bonito Human
Blood 2.6
* The first number refers to the number of passages in hamsters and the second to the
number of in vitro passages.
29
TABLE 2 –LD
50
of pathogenic Leptospira strains
Species Serogroup Strain
NP LD
50
( ± SD)
H C Females Males
L. interrogans Icterohaemorrhagiae
L1-130 4 7 120 (26) 30 (9)
L. interrogans Canicola Kito 2 5 2.4 (1) 2.7 (1)
L. noguchii Bataviae Cascata 2 5 56 (32) 34 (13)
L. noguchii Australis Hook 4 5 10 (1) 100 (1)
L. noguchii Autumnalis Bonito 2 6 1.7 (1) 3.1(1)
NP = Number of passage / H = hamster model / C= culture
SD = Standard deviation
30
TABLE 3 – Outcomes of clinical signs and days until the death in all experiments
Parameters
Species / Strains
L. interrogans L. noguchii
Clinical signs (%) L1-130 Kito Isoton Skoll Bonito Hook Cascata
external hemorrhage 30 30 0 0 0 0 30
dehydration 70 60 0 0 40 40 60
ruffled hair coat 20 10 0 0 10 10 10
Intervals of days until
death
8 - 14 7 - 13 NA NA 7 - 14 10 - 18 11 – 22
Variance 12 10 NA NA 11 13 14
NA = Not applicable
31
FIGURE CAPTIONS
Fig.1. Kaplan-Meier analysis of the results from representative LD
50
experiments with
5 virulent and 2 avirulent strains. Eight hamsters per group were inoculated with a
series of leptospires (10
5
,
■
; 10
4
,
♦
; 10
3
,
▲; 10
2
,
●
; 10
1
,
▼
and 10
0
,
□
) and survivors
were followed for up to 28 days post infection.
Fig. 2. Typical lesions of leptospirosis in a 9 week old hamster dying eight days after
the infection by strain FIOCRUZ L1-130. Marked cell swelling of epithelial cells of
proximal tubules (A, hematoxylin-eosin, 400x). Diffuse loss of cohesion (liver-plate
disarray) of liver cells (B, hematoxylin-eosin, 400x). Microscopic foci of pulmonary
hemorrhage (C, hematoxylin-eosin, 200x).
32
FIGURE 1
Fiocruz L1-130 HR 4.7
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
0
10
20
30
40
50
60
70
80
90
100
Percent survival
Days post inoculation
Kito HR 2.5
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
0
10
20
30
40
50
60
70
80
90
100
Percent survival
Days post inoculation
Cascata HR 2.5
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
0
10
20
30
40
50
60
70
80
90
100
Percent survival
Days Post inoculation
Hook HR 4.5
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
0
10
20
30
40
50
60
70
80
90
100
Percent survival
Days post inoculation
Bonito HR 2.6
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
0
10
20
30
40
50
60
70
80
90
100
Percent survival
Days post inoculation
Isoton/Skoll Strain
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
0
10
20
30
40
50
60
70
80
90
100
Percent survival
Days post inoculation
33
FIGURE 2
34
3. ARTIGO 2
THE TERMINAL PORTION OF LEPTOSPIRAL IMMUNOGLOBULIN-LIKE
PROTEIN LigA CONFERS PROTECTIVE IMMUNITY AGAINST LETHAL
INFECTION IN THE HAMSTER MODEL OF LEPTOSPIROSIS
(Artigo Publicado no Periódico Vaccine)
35
THE TERMINAL PORTION OF LEPTOSPIRAL IMMUNOGLOBULIN-LIKE
PROTEIN LigA CONFERS PROTECTIVE IMMUNITY AGAINST LETHAL
INFECTION IN THE HAMSTER MODEL OF LEPTOSPIROSIS
Éverton F. Silva
a,b
, Marco A. Medeiros
c
, Alan J. A. McBride
a
, Jim Matsunaga
d,e
,
Gabriela S. Esteves
c
, João G. R. Ramos
a
, Cleiton S. Santos
a
, Júlio Croda
a
, Akira
Homma
c
, Odir A. Dellagostin
b
, David A. Haake
d,e
, Mitermayer G. Reis
a
, and
Albert I. Ko
a,f
a
Gonçalo Moniz Research Center, Oswaldo Cruz Foundation, Brazilian Ministry of
Health, Salvador, Brazil
b
Biotechnology Centre, Federal University of Pelotas, Pelotas, Brazil
c
Bio-Manguinhos, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Rio de
Janeiro, Brazil
d
Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California
e
Department of Medicine, the David Geffen School of Medicine at UCLA, Los
Angeles, California
f
Division of International Medicine and Infectious Disease, Weill Medical College of
Cornell University, New York, USA
Corresponding author: Dr. Albert I. Ko, Centro de Pesquisas Gonçalo Moniz,
Fundação Oswaldo Cruz, Rua Waldemar Falcão, 121, Candeal, Salvador, 40296-
710, Bahia, Brasil., Tel. +55 71 3176 2302; fax +55 71 3176 2281., E-mail address:
36
ABSTRACT
Subunit vaccines are a potential intervention strategy against leptospirosis,
which is a major public health problem in developing countries and a veterinary
disease in livestock and companion animals worldwide. Leptospiral immunoglobulin-
like (Lig) proteins are a family of surface-exposed determinants that have Ig-like
repeat domains found in virulence factors such as intimin and invasin. We expressed
fragments of the repeat domain regions of LigA and LigB from Leptospira interrogans
serovar Copenhageni. Immunization of Golden Syrian hamsters with Lig fragments in
Freund’s adjuvant induced robust antibody responses against recombinant protein
and native protein, as detected by ELISA and immunoblot, respectively. A single
fragment, LigANI, which corresponds to the six carboxy-terminal Ig-like repeat
domains of the LigA molecule, conferred immunoprotection against mortality (67-
100%, P <0.05) in hamsters which received a lethal inoculum of L. interrogans
serovar Copenhageni. However, immunization with this fragment did not confer
sterilizing immunity. These findings indicate that the carboxy-terminal portion of LigA
is an immunoprotective domain and may serve as a vaccine candidate for human
and veterinary leptospirosis.
Keywords: Leptospirosis; subunit vaccine; Leptospiral immunoglobulin-like protein;
recombinant protein; immunity; antibodies; hamsters
37
1. INTRODUCTION
Leptospirosis, a spirochetal disease, is a major public health problem
worldwide. The disease is considered to be the most widespread zoonosis in the
world [1,2] due to the pathogen’s ability to induce chronic carriage in the kidney
tubules of a wide range of wild and domestic animals [1,3,4]. Transmission to
humans occurs during direct contact with animal reservoirs or an environment
contaminated by their urine. Infection in 5-15% of the clinical infections causes life-
threatening manifestations such as acute renal failure and pulmonary haemorrhage.
Fatality among severe cases is more than 5-40% [4,5]. Leptospirosis is recognized to
be an emerging infectious disease in developed countries due to outbreaks
associated with sporting events [6] and adventure tourism [7-9], and the increasing
number of cases found among travelers [10], participants of recreational activities
[11] and inner-city populations [12]. However, leptospirosis imparts its greatest
disease burden in developing countries [13]. More than 500,000 cases are reported
each year [2], of which the majority occur among rural subsistence farming
populations [1,3,4] and urban slum dwellers [14-16]. Current control measures have
been uniformly ineffective in addressing leptospirosis in these settings [13,16].
Vaccines represent a potentially cost-effective approach to preventing
neglected tropical diseases, such as leptospirosis, and promoting poverty reduction
[17]. An effective leptospirosis vaccine would conceivably prevent human disease
through immunization of at risk populations or blockade of transmission through
immunization of animal reservoirs. Leptospirosis is an important veterinary health
problem in domestic cattle, pigs and dogs [1, 4,18]. Commercially available vaccines,
consisting of heat or chemically inactivated leptospires, protect hamsters from lethal
infection although protection from sub-lethal infection of the kidneys is incomplete
[19,20]. Yet despite widespread vaccination with whole-cell inactivated vaccines,
leptospirosis remains prevalent in domestic animal populations [4,21]. Several
problems with current vaccine approaches limit their use in humans. Whole-cell
vaccines produce only short-term immunity, requiring administration semi-annually.
Both residual media components and leptospiral lipopolysaccharride (LPS) have
been associated with adverse reactions [1,3,4]. The variability of the LPS
carbohydrate epitopes accounts for the serovar specificity of LPS-based vaccines;
38
there is little cross-protection against infection with the vast majority of other
leptospiral serovars [22-24].
Outer membrane proteins (OMPs) are attractive alternatives to whole-cell
inactivated vaccines because of their antigenic conservation across leptospiral
species and serovars. A number of transmembrane and lipoprotein OMPs have been
shown to be surface-exposed and expressed during infection of the mammalian host
[13,25]. In the form of purified recombinant proteins, the porin OmpL1 and the
lipoproteins LipL41 and LipL32, also known as hemolysis-associated protein 1, have
not been found to be immunoprotective [26,27]. However, when expressed as
membrane proteins in E. coli, OmpL1 and LipL41 exhibit synergistic
immunoprotection in the hamster model of leptospirosis [27]. Immunization of gerbils
with an adenovirus construct encoding LipL32 improved survival to 87% after
challenge with serovar Canicola compared to 51% survival in control-immunized
gerbils [26]. More recently, immunization of gerbils with a pcDNA3.1 DNA vaccine
construct containing the lipL32 gene has also been found to provide partial protection
from lethal challenge [28].
The genes encoding the leptospiral immunoglobulin-like (Lig) repeat proteins
were discovered by screening bacteriophage lambda expression libraries with human
and equine leptospirosis sera [29-32]. The Lig proteins belong to a family of bacterial
immunoglobulin-like (Big) repeat domain proteins that includes intimin and invasin,
the host colonization factors expressed by enteropathogenic E. coli and Yersinia
spp., respectively. Three Lig proteins have been described, designated LigA, LigB,
and LigC. LigA consists of 13 Ig-like imperfect tandem repeats, while LigB and LigC
have 12 Ig-like tandem repeats followed by large ∼80 kDa carboxy-terminal domains
that do not contain Ig-like repeat domains. Virulent forms of L. interrogans serovar
Copenhageni and L. kirschneri serovar Grippotyphosa express LigA and LigB with
sequence-identical amino-terminal regions, while in both strains the locus encoding
LigC is a pseudogene [30]. A mouse-adapted strain of L. interrogans serovar Manilae
expresses LigA and a truncated version of LigB which includes the tandem Ig-like
repeat domains but not the large carboxy-terminal non-repeat domain [29].
Lig proteins are surface-associated moieties [30] and may serve as targets for
bactericidal responses. Recently, Lig proteins have been shown to bind fibronectin
[33], indicating that they may serve as adhesins. Immunization with Lig proteins may
conceivably induce pathogenesis-blocking responses. Kozumi et al demonstrated
39
that immunization of C3H/HeJ mice, which are genetically deficient of to ll-like
receptor 4 [34], with either form of L. interrogans serovar Manilae-derived LigA
protected against lethal challenge [29]. However, mice are significantly less
susceptible to leptospiral challenge than hamsters, gerbils or guinea pigs, which are
the generally accepted animal models for leptospirosis [4]. More recently,
Palaniappan et al evaluated the immunoprotective role of recombinant LigA protein in
hamsters and found that all LigA-immunized animals survived infection with L.
interrogans serovar Pomona [35]. However, 57-88% of the control-immunized
animals survived, which received the same infecting dose (10
8
bacteria) indicating
that the challenge strain was of low virulence. Furthermore, the study did not have
the statistical power to demonstrate that LigA immunization conferred significantly
improved survival in independent experiments. Therefore there is not as of yet,
sufficient evidence to conclude that recombinant Lig proteins confer protection in the
hamster model.
In this study, we produced recombinant Lig protein fragments and
characterized the immune response induced by immunization with these fragments in
hamsters. We found that a LigA fragment conferred protection against lethal
challenge in an infection model that used a highly virulent L. interrogans strain (LD50,
45 bacteria) and showed that the carboxy-terminal unique region of LigA,
corresponding to the last six Ig-like repeat domains, contained an immunoprotective
domain. To our knowledge, this is the first conclusive evidence demonstrating that
immunization with a purified, recombinant protein confers protection in the standard
golden Syrian hamster model for leptospirosis.
2. MATERIAL AND METHODS
2.1. Leptospira strains and serum samples
L. interrogans serovar Copenhageni strain Fiocruz L1-130, isolated from a
patient during an outbreak of leptospirosis in the city of Salvador, Brazil [14,36], was
cultivated in Ellinghausen-McCullough-Johnson-Harris (EMJH) liquid medium (Difco
Laboratories) at 29°C. Culture growth was monitored by counting in a Petroff-
Hausser chamber (Fisher) and dark-field microscopy as described [4]. The clinical
isolate was passaged four times in hamsters and three times in vitro. Seed lots were
then prepared and stored in 25% glycerol at -70°C. Frozen aliquots were thawed and
40
passaged a further four times in EMJH liquid medium prior to use in experiments to
determine the LD50 in hamsters and challenge experiments. Convalescent serum
samples were obtained from patients with laboratory-confirmed leptospirosis during
hospital-based surveillance in Salvador, Brazil [14]. Sera from healthy control
individuals were donated by the state blood bank. Leptospirosis was confirmed by
the microagglutination test (MAT) as previously described [4,37]. The use of subject
sera for these experiments was approved by the Internal Review Boards of the
Oswaldo Cruz Foundation and New York Presbyterian Hospital.
2.2. Cloning, expression and purification of recombinant 6× His tagged Lig
proteins
The proteins LigA and LigB (Fig. 1) were identified as previously described
[30]. The LigANI fragment of LigA, corresponding to nucleotides 1873-3675 of the
ligA sequence (GenBank accession number NC_005823 Region: 533414..537088),
the LigBNI fragment of LigB, corresponding to nucleotides 1873-3773 of the ligB
sequence and the LigBrep fragment of LigB, corresponding to nucleotides 391-1948
of ligB (GenBank accession number NC_005823 Region: 526395..532067), were
selected for expression as recombinant proteins. PCR was used to amplify the target
sequences from genomic DNA purified from L. interrogans Copenhageni Fiocruz L1-
130 with the following primer pairs, LigANI-F 5′- CAATTAAAGATCGTTATACGATAC,
LigANI-R 5′- GGTCTAGATTATGGCTCCGTTTTAATAGAGG; LigBNI-F 5′-
CACCTCCTCTAATACGGATATT, LigBNI-R 5′-TTACACTTGGTTTAAGGAATTAC;
LigBrep-F 5′-ATGGGACTCGAGATTACCGTTACACCAGCCATT, LigBrep-R 5′-
ATTCCATGGTTATCCTGGAGTGAGTGTATTTGT. The resulting 1,802 bp (LigANI)
1,900 bp (LigBNI) and 1,558 bp (LigBrep) PCR products were cloned into the
plasmid pET100-TOPO (Invitrogen) for expression of Lig recombinant proteins with
an N-terminal 6× His tag. All plasmid constructs were confirmed by DNA sequencing
with an ABI 3100 sequencer (Applied Biosystems). E. coli BL21(DE3)Star
transformants containing the Lig constructs were cultured at 37°C to mid log phase
and expression was induced by isopropyl-β-D-thiogalactopyranoside (IPTG), 1 mM
final concentration. The cells were harvested by centrifugation, resuspended in
column buffer (8 M urea, 100 mM Tris, 300 mM NaCl, 5 mM imidazole, pH 8.0), and
disrupted by sonication (3× 1 min pulses; Sonics & Material Inc). The soluble fraction
was recovered (10,000 × g, 10 min) and loaded onto Ni2+-charged chelating
41
sepharose columns (Qiagen). Columns containing bound protein were washed with
10 volumes of column buffer, then 6× wash buffer (6 M urea, 100 mM Tris, 300 mM
NaCl, pH 8.0) containing 5 mM imidazole for the first three washes and increasing to
10 mM imidazole for the remaining washes. His-tagged proteins were eluted from the
column with wash buffer containing 250 mM imidazole. An extended stepwise
dialysis procedure was used to remove urea and imidazole and to promote protein
refolding of the recombinant fragments. Dialysis was performed in 18 steps over a
period of 6 days at 4°C with 100 mM Tris, 300 mM Na Cl, pH 8.0 buffer that contained
decreasing concentrations of urea (6 M to 0 M) in each step. After the stepwise
procedure, the purified protein fragment was dialyzed against phosphate-buffered
saline (PBS, pH 7.2) at 4°C for 24 h and stored at -20°C until use. The Bradford
assay (Bio-Rad) was used to determine protein concentration of purified
preparations.
2.3. Protein gel electrophoresis and immunoblotting of recombinant proteins
For one dimensional sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDSPAGE), samples were solublized in sample buffer (62.5 mM
Tris hydrochloride (pH 6.8), 10% glycerol, 5% 2-mercaptoethanol, 2% SDS) and
separated on a discontinuous buffer system (Mini Protean 3, Bio-Rad). Proteins were
transferred to nitrocellulose membranes following the manufacturer’s instructions
(Mini transblotter, Bio-Rad). Membranes were incubated in blocking buffer (0.05 M
TBS (pH 7.4), 0.05% (v/v) Tween 20 (TBST), 5% (w/v) non-fat dried milk) overnight
at 4°C. After washing in TBST (3× 5 min per wash), membranes were incubated with
sera (diluted 1:200 in TBST) from leptospirosis patients and healthy control
individuals for 1 h at room temperature. Membranes were washed with TBST (4× 5
min per wash) and incubated for 1 h at room temperature with anti-human IgG
conjugated to alkaline phosphatase (Sigma-Aldrich), which was diluted 1:10,000 in
TBST. Membranes were washed in TBST (3× 5 min per wash) and TBS before
colour development using an NBT/BCIP solution following the manufacturer’s
instructions (Bio-Rad).
2.4. Hamster immunization
Female Golden Syrian hamsters with 4 to 5 weeks of age were immunized
subcutaneously with Lig recombinant protein fragments in Freund’s complete
42
adjuvant on day 0 and a second immunization of antigen in Freund’s incomplete
adjuvant on day 14. Immunization was performed with a range of recombinant
protein doses that included 80 µg/40 µg (first/second immunization); 60 µg/30 µg 40
µg/20 µg; and 20 µg/10 µg. Emulsions were prepared by mixing protein fragments
preparations in 200 - 400 µl of PBS with an equal volume of Freund’s adjuvant.
Hamsters were immunized with a maximum of 200 µl per injection site. A negative
control group of hamsters were immunized with an emulsion of Freund’s adjuvant
and PBS. Pre and post-immunization serum samples were collected by phlebotomy
of the retro-orbital venous plexus on the day before the first immunization and on the
day before challenge, respectively. All animal studies were approved by the
Committee for the Use of Experimental Animals of the Oswaldo Cruz Foundation.
2.5. Recombinant Lig ELISA
A preliminary checkerboard analysis was performed to identify the optimal
antigen concentrations and dilutions of hamster sera and antibody conjugate for the
recombinant Lig protein fragment ELISA. The final protocol was based on the
following conditions. Microtitre plates (Costar) were coated with 100 ng of
recombinant Lig protein in 0.1 M sodium carbonate buffer (pH 9.6) at 4°C overnight.
The plates were washed three times with PBS, 0.05% Tween 20 (PBST), and
incubated with 100 µl of blocking buffer (PBST, 1% BSA) at 37°C for 1 h . After
washing with PBST, wells were incubated with hamster serum, diluted 1:100 to
1:25,600 in blocking buffer, at 37°C for 1 h. After washing three times with PBST,
wells were incubated with rabbit anti-hamster IgG conjugated to horseradish
peroxidase (Jackson Immunoresearch Laboratories), diluted 1:25,000, for 37°C for 1
h. After a final cycle of washes (two times with PBST and one time with PBS), 100 µl
of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added to each well. The colour
reaction allowed to develop for 15 min and stopped with the addition of 25 µl of 2 M
H2SO4. Absorbance was determined at 450 nm with a microplate reader (Model 550,
Bio-Rad). Mean values were calculated from serum samples assayed in duplicate.
Each ELISA experiment was repeated three times. Geometric mean end-point titres
(GMT) were determined by linear regression of the OD450 values from a serum
titration to obtain a titre at the intersection with the background OD [38].
2.6. Immunoblotting of native Lig proteins
43
Late log phase cultures of L. interrogans Fiocruz L1-130 were incubated in
EMJH medium supplemented with 120 mM NaCl to induce LigA and LigB expression
as previously described [39]. Leptospires were harvested, washed in PBS,
resuspended in sample buffer and boiled for 10 min prior to SDS-PAGE analysis.
Membranes of immunoblotted extracts were incubated with sera (diluted 1:200) from
hamsters immunized with recombinant Lig proteins for 1 h at room temperature and
after washing were incubated with Rabbit anti-hamster IgG secondary (diluted
1:5,000; Sigma-Aldrich) for 1 h at room temperature. Membranes were incubated
with goat anti-rabbit IgG conjugate (alkaline phosphatase; Jackson Immunoresearch
Laboratories), diluted 1:10,000 for 1 h at room temperature and developed as
described in Section 2.3.
2.7. Hamster challenge studies
Challenge experiments were performed with nine-week-old hamsters in
groups of eight to determine the 50% lethal dose (LD50) of L. interrogans
Copenhageni Fiocruz L1-130. Hamsters were challenged with an inoculum of 10
0
-
10
5
leptospires, diluted in PBS and administered intraperitoneally. Hamsters were
monitored three times a day during the 28 day post-challenge period and euthanized
when clinical signs of terminal disease appeared. The LD50 was calculated by the
method of Reed-Muench [40]. For vaccine protection experiments, groups of 6-10
hamsters, immunized according to protocols described in section 2, were challenged
at age 7 to 9 weeks with an intraperitoneal administration of 10
3
leptospires seven
days after the second immunization. Hamsters were monitored daily for clinical signs
of leptospirosis and euthanized when clinical signs of terminal disease appeared.
Surviving hamsters on day 28 post-challenge were euthanized. Blood, urine, kidney,
lung and liver tissues were harvested for serological, culture, and histopathology
studies. Sterilizing immunity was determined based on culture isolation of
leptospires, identification of leptospirosis-associated pathology and histological
detection of leptospires in tissues of surviving hamsters. Kidney tissue and blood
samples were used to inoculate EMJH medium. Dark-field microscopy was
performed during an 8 week incubation period to identify positive cultures. Tissue
sections were stained with hematoxylin and eosin for evidence of interstitial nephritis,
pulmonary haemorrhage and liver diffuse dissociation. Warthin-Starry silver staining
was performed to identify leptospires in tissues [41]. A negative control group of
44
hamsters were immunized with an emulsion of PBS and Freund’s adjuvant and
inoculated with a lethal inoculum dose of leptospires according to the same protocol
as described for recombinant Lig protein-immunized hamsters. A positive control
group of hamsters were immunized with killed whole-leptospires. Washed pellets of
cultures L. interrogans strain Fiocruz L1-130 were heat-inactivated 56°C fo r 20 min,
resuspended in PBS and stored at -20°C until use. H amsters were immunized with a
dose of 10
9
inactivated leptospires on day 0 and day 14 and challenged on day 28.
2.8. Statistical analysis
The Students t-test was used to determine significant differences between the
geometric mean titres obtained in ELISA results. The Fisher Exact test and log-rank
sum test were used to determine significant differences for mortality and survival,
respectively, among the groups immunized with Lig protein fragments and the
negative control group. The chi-squared test for trend was used to evaluate
significant differences between hamster groups immunized with different doses in the
immunoprotection experiments. All P values were two-sided and a P value of <0.05
was considered to indicate statistical significance. EpiInfo 6.04d (Centers for Disease
Control and Prevention) and GraphPad Prism 4 software systems (GraphPad
Software) were used to perform the statistical analyses.
3. RESULTS
3.1. Preparation of purified recombinant Lig proteins
Three protein fragments were cloned and expressed comprising 90% and 60%
of the entire LigA and LigB molecules, respectively (Fig. 1). The LigBrep construct
spans the 2nd to 7
th
Big repeat domains of the LigA and LigB, a region of sequence
identity between the two proteins. The LigANI construct spans the 7th to 13th Big
repeat domains of the LigA molecule. The LigBNI construct spans the 7th to 12th Big
repeat domains of the LigB molecule. The sequence identity between these two non-
identical regions is 38%. Cloning of three PCR products, corresponding to LigANI,
LigBNI and LigBrep, in pET100-TOPO expression vector allowed for purification of 6-
10 milligrams of His-tagged recombinant protein per litre of transformed E. coli host
culture. However, the three Lig recombinant fragments were expressed as inclusion
bodies and required denaturing conditions for purification, followed by prolonged
45
stepwise dialysis to obtain soluble protein preparations. Protein fragment
preparations were obtained which had over 95% purity (Fig. 2A) and reacted with
pooled leptospirosis patient sera in immunoblot analysis (Fig. 2B).
3.2. Antibody response induced by immunization with recombinant Lig protein
fragments
Golden Syrian hamsters were immunized with the three Lig protein fragments
in Freund’s adjuvant. Sera obtained seven days after the 2nd immunization from
LigANI and LigBNI immunized hamsters demonstrated high titre binding to
homologous recombinant Lig fragments in an ELISA (Fig. 3A and 3B). Absorbance
values for immune sera were significantly greater than pre-immune sera at titres up
to 1:25,600. Although immunization with LigBrep produced specific antibodies that
recognized the homologous recombinant fragment, absorbance values for immune
sera were not significantly greater than those of preimmune sera for titres >1:1,600
(Fig. 3C). Absorbance values for serum samples from hamsters immunized with PBS
and Freund’s adjuvant were <0.06 when diluted 1:100 in ELISAs with the three
recombinant fragments (data not shown). Immunization with a specific Lig fragment
produced significant cross-reactive antibody responses against the other two
fragments. GMTs ranged from 1:1,600 to 1:25,600 in ELISAs that measured cross-
reactive antibodies in sera from immunized hamster to heterologous Lig proteins
(data not shown). Recombinant protein-based ELISA analysis was performed with
sera from hamsters immunized with the LigANI fragment to determine the duration of
humoral immune response and the response conferred by a range of immunization
doses (1st/2nd doses: 20/10, 40/20 and 80/40 µg, Fig. 4). Hamsters produced an
antibody response with GMTs of 1:500-1:1,500 one week after primary immunization
with 20-80 µg of recombinant LigANI. GMTs peaked one week after the second
immunization with 10-40 µg of recombinant LigANI (GMT range, 1:8,000-1:29,000)
and remained elevated (>1:3,000) until the last observation point, 63 days after
primary immunization. Of note, the standard deviation was large (1:4,600 to 1:8,700)
for sera collected at the 28-day time point from hamsters immunized with 20/10 µg of
recombinant fragment due to high end-point titres in one animal (1:18,000).
Immunization doses with 80/40 µg (1st/2nd immunization doses) produced
significantly higher GMT at all time points in comparison to 40/20 and 20/10
µg
immunization schemes.
46
3.3. Immunization with recombinant Lig proteins induces antibodies that
recognize native Lig proteins
Immunoblot analysis of sera from groups of four hamsters found that
immunization with each of the recombinant Lig proteins produced antibodies that
recognize native Lig proteins in whole Leptospira lysates (Fig 5). Sera from LigBrep
immunized hamsters reacted with LigA and LigB proteins while sera from LigANI-
immunized hamsters reacted specifically with LigA. Of note, sera from one of four
LigBNI immunized hamsters recognized native LigB, yet all reacted with native LigA.
3.4. Determination of LD50 for the L. interrogans challenge strain and
characterization of the hamster infection model
A series of four challenge experiments found that the mean LD50 for L.
interrogans serovar Copenhageni strain Fiocruz L1-130 was 45.9 (± 9.3) leptospires
in nine week-old hamsters (Table 1). Death occurred 10-18 days after challenge and
occurred significantly earlier in groups that received higher challenge doses. A
challenge dose of 10
3
leptospires (∼20x LD50) induced death in all hamsters, which
occurred between the 10th-18th day post-challenge (Fig. 5). Hamsters developed the
characteristic clinical and histopathologic signs of leptospirosis. At the time of death,
hamsters were jaundiced and demonstrated bleeding diatheses. Autopsies of
infected hamsters found interstitial nephritis, acute damage of tubular epithelia,
marked dissociation of liver trabecula with hepatocytes presenting reactive changes
such as cytoplasmic size variation, prominent nucleoli and binucleation, and
pulmonary haemorrhage as primary pathological findings. Hamsters are a standard
model used to evaluate protective immunity elicited by whole-cell vaccines [20].
Immunization with whole Leptospira-based preparations conferred protection (100%)
against lethal challenge (10
3
leptospires) with the Fiocruz L1-130 strain (Table 2),
indicating that the study’s experimental model reproduced findings reported for
established models of vaccine-based immunity.
3.5. Protection conferred by immunization with recombinant Lig protein
fragments
Evaluation of recombinant Lig proteins in the hamster model of vaccine-
induced immunity found that immunization with the LigANI fragment in Freund’s
adjuvant conferred protection against lethal challenge with 10
3
leptospires (20x
47
LD50) (Fig. 6A and Table 2). Protection conferred with immunization of 80/40 µg
(1st/2nd immunization dose) of LigANI ranged from 67 to 100% in the six
experiments in which four different batches of recombinant protein were used. There
were no significant differences between protection levels between these
experiments. Immunization with the LigBrep and LigBNI fragments did not induce
protection with respect to either mortality or survival. LigANI-immunized hamsters did
not show clinical evidence of infection during the 28 day follow-up period after
challenge infection. Although autopsy examination did not find macroscopic or
histological evidence for disease in LigANI immunized hamsters that survived lethal
challenge and were sacrificed 28 days post-challenge infection, leptospires were
isolated and/or detected in kidney tissues. In contrast, leptospires were not isolated
and/or detected in tissues of hamsters immunized with whole Leptospira preparations
and infected with a lethal dose of leptospires, indicating that the whole-cell vaccine
conferred sterile immunity. Immunization regimens with lower doses (1st/2nd doses:
20/10, 40/20 and 60/30 µg) of LigANI fragment conferred a level of protection (63-
88%) against lethal challenge similar to the initial 80/40 µg regimen (Table 2). The
level of vaccine-induced protection was not significantly different between groups
immunized with different immunization doses. Among LigANI immunized hamsters
that died after lethal infection, death occurred significantly later than for the control
group that received a lethal challenge dose (Fig. 6B). Pre-challenge sera were
analyzed from randomly selected LigANI-immunized hamsters in the six experiments
(Table 2) to determine whether differences in antibody responses correlated with
outcome after lethal challenge. All pre-challenge sera from LigANI-immunized
hamsters recognized native LigA protein in immunoblot analysis of whole Leptospira
extracts, regardless of the outcome. In recombinant LigANI-based ELISA, mean
GMTs of pre-challenge sera (1:8,000) from hamsters that died were lower, albeit not
significantly different, than the mean GMTs (1:17,000) of sera from surviving
hamsters.
4. DISCUSSION
We have previously shown that Lig proteins are surface-exposed outer
membrane proteins of virulent leptospires [30] and therefore potential targets of a
protective immune response. Previous studies examined the ability of Lig proteins to
48
confer immunoprotection, yet the conclusions that could be drawn were limited
because of the animal models and low virulence of the challenge strains used.
Kozumi et al demonstrated that immunization with recombinant full-length LigA and
truncated LigB proteins protected C3H/HeJ mice against lethal infection [29]. These
findings were the first to suggest that Lig proteins are immunoprotective moieties.
However, the mouse is not an ideal animal model of leptospirosis because it is
relatively resistant to challenge with pathogenic Leptospira strains and requires a
high inoculum (>10
6
bacteria) to produce lethal infection [42,43]. Palaniappan et al
used hamsters, an accepted model for leptospirosis, to evaluate LigA protein-induced
immunoprotection but did not demonstrate a significant difference in survival between
LigA and control-immunized animals in independent experiments due to low mortality
rates (12-43%) among control-immunized animals [35].
In this study, we evaluated a panel of recombinant Lig protein fragments in the
hamster infection model and found that the carboxy-terminal region unique to the
LigA protein (LigANI) contained an immunoprotective domain. Fiocruz L1-130, a
highly virulent strain of L. interrogans serovar Copenhageni was used for which a low
inoculum dose (10
3
bacteria, 20x LD50) reproducibly induced characteristic disease
manifestations and death in all control immunized animals. LigANI conferred an
average level of vaccine efficacy (Table 2) which is the highest reported for a subunit
vaccine in the hamster model.
This study provides evidence that a single purified recombinant protein is able
to induce protective immunity against lethal challenge in the standard hamster model
of leptospirosis. Previous studies using purified recombinant proteins, including
OmpL1, LipL41 and LipL32 [26,27], did not demonstrate immunoprotection in
hamsters, even though these proteins have been shown to contain immunoprotective
epitopes using other approaches [27,28]. Evidence for LipL32 as a protective antigen
comes from studies involving immunization with recombinant adenovirus [26] and
naked plasmid DNA constructs [28]. The most likely explanation for the lack of an
immunoprotective effect with recombinant proteins is the failure of the proteins to fold
correctly and recapitulate their native secondary structure. Structural modelling of Lig
molecules predicts that the repeat domains have a highly folded β-immunoglobulin
sandwich structure [30]. Lig protein fragments used in this study were purified under
denaturing conditions (8 M urea), and allowed to renature gradually through a
stepwise dialysis method of urea removal. This approach may have led to the correct
49
refolding of some but not all of the critical conformational epitopes present in the Lig
molecules, indicating a need for further refinement of the recombinant protein
preparation protocol.
The mechanism of LigANI vaccine-mediated immunity remains speculative at
this point. Immunization with LigANI produced robust antibody responses to
recombinant and native protein at the time at which animals were challenged. Since
LigA is a surface-exposed moiety, anti-LigA antibodies may have bactericidal or
opsonophagocytic activity. Of note, the carboxy-terminal unique repeat domain
region of LigA (LigANI) and LigB (LigBNI) has recently been shown to have
fibronectin binding activity [33]. Anti-LigANI antibodies may therefore conceivably
block key pathogen-host cell interactions in disease pathogenesis.
In this study, anti-recombinant LigA antibody titres were lower in pre-challenge
sera from LigANI-immunized hamsters that died in comparison to those that survived
after lethal infection, although this difference was not significant. We interpret this
result as an indication that the recombinant LigA protein fragment used to immunize
hamsters contains both protective and non-protective epitopes. Elucidation of the role
of the anti-LigA antibody response requires determination whether protective
immunity can be passively transferred. Alternatively, LigA may induce protective cell-
mediated responses as has been observed with whole-cell vaccines in cattle [44]. Of
note, computational analyses found that LigA contains predicted T-cell epitopes [45].
Whole-cell inactivated leptospiral vaccines were developed by Ido and
colleagues in the early 20th century [46] and have widespread commercial availability
for livestock and companion animals. These whole-cell vaccines provide serovar-
specific protection against accidental infection [4,13]. The lack of serovar cross-
protection, the need for annual or biannual revaccination, and a high frequency of
side effects are serious disadvantages that have limited the availability of whole-cell
Leptospira vaccines for human use. A well-defined subunit vaccine composed of a
cross-protective antigen that results in long-term immunity would be of great interest
in the prevention of human leptospirosis.
Several challenges remain to be addressed to develop a subunit vaccine
based on recombinant Lig proteins. In this study, immunizations were performed with
Freund’s adjuvant. The efficacy of Lig protein formulations must be determined using
adjuvants acceptable for human use, such as aluminium hydroxide. An ideal
recombinant protein-based vaccine formulation would comprise a single fragment
50
that confers cross-protective immunity against infection with heterologous serovars.
Full-length ligA gene sequences have been identified in a range of L. interrogans and
L. kirschneri serovars [29-32,36]. The predicted amino acid sequence homology is
80-97% for LigA proteins from these serovars [30]. Although ligA was not identified in
the genome sequences of L. borgpetersenii serovar Hardjo [47] and L. interrogans
serovar Lai [48], individual Big domains in LigA and LigB proteins share an amino
acid sequence identity of 17-99% with each other. In this study, immunization with
the LigANI fragment produced cross-reactive antibody responses against LigBrep
and LigBNI fragments, which in turn may have contributed to the protective immunity
observed in the hamster trials. In summary, we have shown that a recombinant
protein fragment of the putative virulence determinant, LigA, reproducibly conferred
high-level protection against lethal infection in the hamster model for leptospirosis.
These findings provide proof-of-concept evidence for the feasibility of developing a
subunit vaccine for human leptospirosis.
ACKNOWLEDGMENTS
This work was supported by the Brazilian National Research Council (grants
300861/1996, 01.06.0298.00 and 420067/2005, and 554788/2006); Bio-Manguinhos,
Oswaldo Cruz Foundation, Brazilian Ministry of Health (grant 09224-7); VA Medical
Research Funds; the National Institute of Allergy and Infectious Diseases (grants
AI052473 and AI034431) and Fogarty International Centre (grant TW00919).
51
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56
TABLE 1. Determination of the LD
50
for L. interrogans strain Fiocruz L1-130 in the
Golden Syrian hamster infection model.
Challenge dose (No. leptospires)
Experiment 10
5
10
4
10
3
10
2
10
1
10
0
LD
50
a
No. deaths per group (N=8)
1 7 8 8 8 ND
b
ND 36.5
2 7 8 8 6 ND ND 56.2
3 8 7 8 5 3 0 39.8
4 8 8 8 5 1 0 51.3
Mean ±SD
c
45.9 ± 9.3
a
Fifty percent lethal dose
b
Not determined
c
Starndard deviation
57
TABLE 2. Protection conferred by immunization with recombinant Lig protein
fragments against lethal challenge in the hamster model.
Experiment
1
st
/2
nd
Immunization
Dose (µg)
LigANI LigBNI LigBrep Bacterin
a
Control
immunization
b
% Protection (No. survivors/total)
1 80/40 80.0 (8/10)
c
10.0 (1/10) 0 (0/10) 100 (4/4)
c
0 (0/10)
2 80/40 88.9 (8/9)
c
0 (0/9) ND 100 (4/4)
c
0 (0/9)
3 80/40 66.7 (6/9)
c
22.2 (2/9) ND ND 0 (0/9)
4 80/40 83.3 (5/6)
c
ND ND ND 0 (0/6)
5 80/40 70.0 (7/10)
c
ND ND ND 0 (0/10)
6
80/40 100 (8/8)
c
ND ND ND 0 (0/8)
60/30 75.0 (6/8)
c
ND ND ND 0 (0/8)
40/20 87.5 (7/8)
c
ND ND ND 0 (0/8)
20/10 62.5 (5/8)
c
ND ND ND 0 (0/8)
ND, not determined.
a
Immunizations were performed by intraperitoneal administration of two doses (10
9
organisms )
of sonicated L. interrogans strain Fiocruz L1-130, separated by two week interval.
b
Control immunizations were performed by administering buffer (PBS) with Freunds adjuvant.
c
Protection against lethal challenge was statistically significant (P <0.05).
58
FIGURE CAPTIONS
Fig 1. Schematic representation of LigA and LigB proteins and recombinant
fragments. Boxes represent bacterial immunoglobulin-like (Big) tandem repeat
domains (∼90 amino acids). Amino acids 102 to 630 (Big domains 2-6 and part of 7)
of LigA and LigB, the region with 100% amino acid sequence identity between these
two proteins, are represented as grey boxes. The C-terminal Big domains of LigA
(amino acid position 631-1,224) and LigB (amino acid position 631-1,119) have lower
amino acid sequence identity (38%) and are represented as hatched boxes. Lines
represent the three recombinant fragments, LigANI, LigBrep and LigBNI that were
cloned and expressed.
Fig 2. Recombinant Lig proteins and seroreactivity with sera from leptospirosis
patients. (A) Coomassie blue-stained SDS-PAGE gel of purified LigANI, LigBNI and
LigBrep recombinant protein fragments (1.5 µg/lane). (B) Immunoblot analyses of
LigANI, LigBNI and LigBrep recombinant protein fragments. Membranes were probed
with pooled convalescent sera from leptospirosis patients (+) and from healthy
individuals (-). Positions of molecular mass markers (kDa) are shown on the left.
Fig 3. Antibody responses in hamsters immunized with recombinant Lig proteins.
ELISA reactions were performed to determine antibody levels against LigANI (A),
LigBNI (B) and LigBrep (C) in sera from hamsters immunized with LigANI (A: pre-
immune, □; immune, ■) LigBNI (B: pre-immune, □; immune, ▴) and LigBrep (C: pre-
immune, □; immune, ▾). Mean absorbance (optical density, 450nm) +/-standard
deviation (bars) are shown for a representative experiment among three that
evaluated sera from groups of four immunized hamsters.
Fig 4. Kinetics of IgG antibody response in hamsters immunized with recombinant
LigANI protein fragment. ELISA was used to determine anti-LigANI IgG antibody
levels in hamsters immunized on day 0 to 14 (arrows) with LigANI doses of 80 and
40
µg (■), 40 and 20 µg (●) and 20 and 10 µg (□), respectively. Normalized geometric
59
mean endpoint titres (GMTs) and standard deviation (bars) for sera collected from
groups of four hamsters at weekly intervals over a 63 day follow-up period are
shown.
Fig 5. Sera from hamsters immunized with recombinant Lig fragments recognize
native LigA and LigB proteins. Immunoblots of whole-cell extracts of L. interrogans
strain Fiocruz L1-130 (10
8
organisms per lane) were probed with representative
samples of pre-immune (PI) and immune sera from hamsters immunized with LigANI,
LigBNI, LigBrep and PBS (Control) with Freund’s adjuvant. The mobility of molecular
mass markers and expected positions of LigA (128 kDa) and LigB (201 kDa) are
shown on the left and right side, respectively of the figure.
Fig 6. Survival of hamsters immunized with recombinant Lig proteins after lethal
challenge. A: Post challenge survival is shown for a representative vaccine
evaluation (Experiment 1, Table 2) in which hamsters were immunized on day -21
(80 µg) and day -7 (40 µg) with LigANI (■), LigBNI (▴), LigBrep (□) and PBS (▾) and
challenged with L. interrogans strain Fiocruz L1-130 on day 0. B. Protective effect
conferred by immunization with increasing LigANI doses (80/40 µg, ■; 60/30 µg, □;
40/20 µg, ▴; 20/10 µg, ●; and 0/0 µg, ▾) on post-challenge survival (Experiment 6,
Table 2).
60
FIGURE 1
61
FIGURE 2
62
FIGURE 3
63
FUGURE 4
64
FIGURE 5
65
FIGURE 6
66
4. ARTIGO 3
VACCINATION WITH A NOVEL LIPOPROTEIN CONFERS PROTECTIVE
IMMUNITY AGAINST LETHAL INFECTION IN THE HAMSTER MODEL OF
LEPTOSPIROSIS
(Artigo que será submetido como Short communication ao periódico Clinical and
Vaccine Immunology)
67
VACCINATION WITH A NOVEL LIPOPROTEIN CONFERS PROTECTIVE
IMMUNITY AGAINST LETHAL INFECTION IN THE HAMSTER MODEL OF
LEPTOSPIROSIS
ABSTRACT
Four leptospiral lipoproteins were selected and evaluated as vaccine
candidates. The coding sequence of these lipoproteins were amplified by PCR from
Leptospira interrogans serovar Copenhageni, cloned and expressed in Escherichia
coli. Immunization of hamsters with purified recombinant lipoproteins revealed that
one of them was able to confer protection against mortality (P <0.001) in hamsters
which were challenged with a lethal dose of a virulent strain. This is the only
recombinant antigen evaluated to date that was able to fully protect hamsters against
leptospirosis when administered with aluminum hydroxide as adjuvant.
Keywords: Leptospirosis; subunit vaccine; recombinant lipoprotein; immunization;
hamsters
Leptospirosis is a bacterial disease that affects both humans and animals (27).
It is found in a wide variety of environmental contexts, in industrialized and
developing countries, and in urban and rural areas (1). The number of human and
animal cases worldwide is not well-documented. During outbreaks and in high-risk
groups, 100 or more per 100,000 may be infected and for several reasons
leptospirosis is overlooked and consequently underreported (26). It is currently
considered a neglected tropical bacterial disease (10). In developing countries,
leptospirosis has become an urban health problem as slum settlements have grown
and outbreaks in these communities have increased (11, 13, 20). In these settings,
current control measures have been ineffective in addressing leptospirosis (15).
Pathogenic Leptospira are highly invasive bacteria capable of infecting a broad range
of mammalian hosts (4), and the host infection produces a diverse array of clinical
manifestations (1). This infecting agent is transmitted from one animal carrier to
another, or to humans, via direct or indirect contact with urine or other body fluids
that contain viable leptospires (14).
68
Current vaccines against leptospirosis target the lipopolysaccharide coat of
leptospires, which has over 230 pathogenic serovars, and many different strains with
small antigenic differences can be found in some serovars, thus limiting cross-
protection (1). The identification of new targets expressed during infection is
important for the development of new immunoprotective strategies (8). To date, there
is incomplete knowledge on the mechanisms of protective immunity against
leptospiral infection, and the current emphasis in research laboratories is to discover
cross-species and cross-serovar-conserved protective antigens that may provide
longer-term protection from a broad range of Leptospira (25)
Several recombinant outer membrane proteins (OMP) have been shown to be
protective immunogens in animal models of bacterial diseases (6, 12, 24).
Recombinant leptospiral OMPs such as OmpL1, LipL41, LipL32, and Leptospiral
immunoglobulin-like protein A (LigA) have been evaluated as potential vaccine
candidates (3, 5, 9, 17). Our group has recently demonstrated that vaccination with a
LigA fragment (22) or with a recombinant Mycobacterium bovis BCG expressing the
LipL32 antigen (21), may constitute potential intervention strategies against
leptospirosis. In the present study we evaluate four lipoproteins previously identified
as potential subunit vaccine candidates (7). Our findings revealed that one of the
lipoproteins provides a significant level of protection against lethal challenge in
hamster model of leptospirosis.
L. interrogans serovar Copenhageni strain Fiocruz L1-130 (13, 16) was
cultivated in Ellinghausen-McCullough-Johnson-Harris (EMJH) liquid medium (Difco
Laboratories) at 29 °C. Culture growth was monitore d by counting in a Petroff-
Hausser chamber (Fisher) and dark-field microscopy as previously described (4).
Escherichia coli strains TOP10F and BL21(DE3)-RIL were grown in Luria-Bertani
(LB) medium at 37 °C (19). Primers were designed to include most of the targeted
genes but not their highly hydrophobic signal sequences. The targets were arbitrarily
named Lip1 to Lip4. Coding sequences were amplified by PCR and cloned into the
expression vector pQE30 (Invitrogen) or pAE (18). Recombinant plasmids were used
to transform E. coli BL21(DE3) strains by electroporation and His-tagged proteins
were purified by affinity chromatography in a nickel (Ni
+2
) charged Sepharose column
using the ÄKTAPrime chromatography system (Amersham Biosciences, USA).
Dialysis procedure was used to remove urea and imidazole and to promote refolding
of the recombinant proteins. Solution containing recombinant proteins (200 mM
69
Na
2
HPO
4
, 0,5 M NaCl, 5mM Imidazole, 0,2% N-Lauroylsarcosine) were dialyzed
against 100 mM Tris-HCl, 150 mM NaCl, pH 8.0 buffer, overnight at 4°C. Protein in
the final preparation were quantified by the Bradford method (2).
Female Golden Syrian hamsters 4-5 weeks old were allocated into groups of 6
animals and immunized by intramuscular route with recombinant protein in aluminum
hydroxide (15%) on day 0 and day 14. Immunization was performed with
recombinant protein doses of 80 µg/40 µg (first/second immunization). Hamsters
were immunized with 250 µL per injection site. A negative control group of hamsters
was immunized with aluminum hydroxide adjuvant and PBS. Pre and post-
immunization serum samples were collected by phlebotomy of the retro-orbital
venous plexus on the day before the first and second immunization, and on the day
before challenge. Immunized hamsters were challenged at age 8-9 weeks, fourteen
days after the second immunization, with an intraperitoneal (i.p.) administration of
100 leptospires (5 × LD
50
) of strain Fiocruz L1-130 (21). Hamsters were monitored
daily for clinical signs of leptospirosis and euthanized when clinical signs of terminal
disease appeared. Surviving hamsters were euthanized on day 24 post-challenge
and the blood, urine, kidney, lung and liver tissues were harvested for serological,
culture, and histopathology studies. Sterilizing immunity was determined as
described (22). Experimental animals were maintained at the animal facility of the
Biotechnology Center of the Federal University of Pelotas (UFPel). The animals were
manipulated in accordance with the guidelines of the Ethics Committee in Animal
Experimentation of the UFPel throughout the experimental period.
The Fisher Exact test and log-rank sum test were used to determine significant
differences for mortality and survival, respectively, among the groups immunized with
recombinant proteins and the negative control group. All P values were two-sided
and a P value of <0.05 was considered to indicate statistical significance. EpiInfo
6.04d (Centers for Disease Control and Prevention) and GraphPad Prism 4 software
systems (GraphPad Software) were used to perform the statistical analyses.
All PCR products were successfully cloned and expressed in E. coli as a
6×His tag N-terminus fusion which allows purification of the proteins by affinity
chromatography. All four proteins were expressed as insoluble inclusion bodies.
They were solubilized and purified using a buffer containing urea. After dialysis and
SDS-PAGE, purity was estimated to be over 95%. The yield of purified protein
ranged from 4 mg/L to 80 mg/L of culture, depending on the protein.
70
Immunization with recombinant proteins in aluminum hydroxide adjuvant
conferred protection against lethal challenge only in the group of animals vaccinated
with Lip1. Protection conferred by immunization with 80/40 µg (1st/2nd immunization
dose) of Lip1 was 100% (P<0.001), whereas only one animal survived in the groups
immunized with Lip2 and with Lip3. No animal survived in the control group, and in
the group immunized with Lip4 (Table 1 and Fig. 1). Hamsters that survived the
challenge did not show clinical evidence of infection during the 24 day follow-up
period after challenge infection. Necropsy examination did not reveal macroscopic or
histological evidence of disease and attempt to isolate leptospires from kidney tissue
failed, indicating that the Lip1 conferred sterilizing immunity. This result provides
evidence that, similarly to LigA fragments, a single purified recombinant lipoprotein
can induce protective immunity against lethal challenge in the hamster model of
leptospirosis. Furthermore, the efficacy of this formulation with Lip1 was
demonstrated using aluminum hydroxide, an adjuvant acceptable for human and
animal use.
The mechanism of immune protection in leptospirosis remains unknown.
There is a need for studies directed towards defining how the immunity resulting from
lipoprotein immunization can be improved. Determination of the relative contributions
of humoral immune mechanisms and cellular-mediated immunity, for protection of the
hamster model, would provide insights about immune response against leptospirosis.
Experiments aiming at evaluating passive protection by sera from immunized rabbits
and hamsters are currently being performed.
Extensive efforts have been made to identify cross-protective antigens of
Leptospira (23, 25). Lipoproteins and other membrane associated proteins are
potential vaccine candidates, however, only a few leptospiral proteins characterized
so far have been able to confer protection when used to immunize susceptible
animals (9, 17, 21-22). LipL32, the major outer membrane protein found in all
pathogenic species of Leptospira, was able to confer partial protection only when
administered as a DNA vaccine or expressed in adenovirus (3) or in recombinant
BCG (21). No protection was observed when the protein was used as a subunit
vaccine administered with aluminum hydroxide adjuvant. In this study we identified a
lipoprotein that when administered with aluminum hydroxide was able to confer full
protection in the hamster model of leptospirosis. Unlike LigA, Lip1 conferred
sterilizing immunity, as no Leptospira could be isolated from kidney tissue 24 days
71
after challenge. Further experiments will have to be performed to confirm this result
and to determine the minimal dose. It is expected that the amount of protein may be
reduced considerably, lowering the cost per dose and making the vaccine affordable
even for human and animal vaccination.
72
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Table 1. Protection conferred by immunization with recombinant lipoproteins against
lethal challenge in the hamster model.
Group
a
Days until death no. survivors/total % Protection
Lip1
- 6/6
100
b
Lip2
11,13,13,13,18 1/6 16.7
Lip3
11,11,11,11,13 1/6 16.7
Lip4
11,11,12,13,14,14 0/6 0
Control
c
11,11,12,13,13,14 0/6 0
a
Animals were vaccinated two times (
80 / 40
µg)
at 2-week interval and were challenged with L1-130
two week after the last vaccination.
b
Protection against lethal challenge was statistically significant (P<0.001).
c
Control immunizations were performed by administering PBS with aluminum hydroxide adjuvant.
77
FIGURE CAPTIONS
Fig 1. Survival of hamsters immunized with recombinant proteins after lethal
challenge. Post-challenge survival is shown for a representative vaccine
evaluation in which hamsters were immunized on day 28 (80 µg) and day 14
(40 µg), and challenged with L. interrogans strain Fiocruz L1-130 on day 0.
78
FIGURE 1
0 2 4 6 8 10 12 14 16 18 20 22 24
0
25
50
75
100
Lip2
Lip3
Lip4
Lip1
Control
Days post challenge
Percent survival
79
5. CONCLUSÕES
- A caracterização da virulência de cepas de Leptospira permite a definição da
LD
50
de cada uma delas, e serve para a avaliação de futuros candidatos vacinais
contra a leptospirose humana e animal.
- O fragmento LigANI induz uma resposta imune humoral significativa (≥
25.600) e duradoura (> 63 dias).
- LigANI protege hamsters contra infecção letal (67-100%), mas não impede a
colonização renal, enquanto que, LigBNI protege parcialmente contra infecção letal
(10-23%) e contra a colonização renal nos hamsters analisados. Quanto aos demais
alvos avaliados.
- Lip1 induz uma resposta imune protetora significativa (P <0.001), e protege
contra a colonização renal.
- LigANI e Lip1 são candidatos potenciais para compor uma vacina
recombinante contra leptospirose humana e animal.
80
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89
7 ANEXO
Artigo 2: Publicado no Periódico Vaccine
Vaccine 25 (2007) 6277–6286
The terminal portion of leptospiral immunoglobulin-like protein
LigA confers protective immunity against lethal infection
in the hamster model of leptospirosis
´
Everton F. Silva
a,b
, Marco A. Medeiros
c
, Alan J.A. McBride
a
, Jim Matsunaga
d,e
,
Gabriela S. Esteves
c
,Jo
˜
ao G.R. Ramos
a
, Cleiton S. Santos
a
,J
´
ulio Croda
a
, Akira Homma
c
,
Odir A. Dellagostin
b
, David A. Haake
d,e
, Mitermayer G. Reis
a
, Albert I. Ko
a,f,∗
a
Gon¸calo Moniz Research Centre, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Salvador, Brazil
b
Biotechnology Centre, Federal University of Pelotas, Pelotas, Brazil
c
Bio-Manguinhos, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Rio de Janeiro, Brazil
d
Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, CA, USA
e
Department of Medicine, The David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
f
Division of International Medicine and Infectious Disease, Weill Medical College of Cornell University, NY, USA
Received 18 April 2007; received in revised form 15 May 2007; accepted 23 May 2007
Available online 14 June 2007
Abstract
Subunit vaccines are a potential intervention strategy against leptospirosis, which is a major public health problem in developing countries
and a veterinary disease in livestock and companion animals worldwide. Leptospiral immunoglobulin-like (Lig) proteins are a family of
surface-exposed determinants that have Ig-like repeat domains found in virulence factors such as intimin and invasin. We expressed fragments
of the repeat domain regions of LigA and LigB from Leptospira interrogans serovar Copenhageni. Immunization of Golden Syrian hamsters
with Lig fragments in Freund’s adjuvant induced robust antibody responses against recombinant protein and native protein, as detected by
ELISA and immunoblot, respectively. A single fragment, LigANI, which corresponds to the six carboxy-terminal Ig-like repeat domains of
the LigA molecule, conferred immunoprotection against mortality (67–100%, P < 0.05) in hamsters which received a lethal inoculum of L.
interrogans serovar Copenhageni. However, immunization with this fragment did not confer sterilizing immunity. These findings indicate
that the carboxy-terminal portion of LigA is an immunoprotective domain and may serve as a vaccine candidate for human and veterinary
leptospirosis.
© 2007 Elsevier Ltd. All rights reserved.
Keywords: Leptospirosis; Subunit vaccine; Leptospiral immunoglobulin-like protein; Recombinant protein; Immunity; Antibodies; Hamsters
1. Introduction
Leptospirosis, a spirochetal disease, is a major public
health problem worldwide. The disease is considered to be
the most widespread zoonosis in the world [1,2] due to the
pathogen’s ability to induce chronic carriage in the kidney
tubules of a wide range of wild and domestic animals [1,3,4].
∗
Corresponding author at: Centro de Pesquisas Gonc¸alo Moniz, Fundac¸
˜
ao
Oswaldo Cruz, Rua Waldemar Falc
˜
ao, 121, Candeal, Salvador, 40296-710
Bahia, Brazil. Tel.: +55 71 3176 2302; fax: +55 71 3176 2281.
Transmission to humans occurs during direct contact with
animal reservoirs or an environment contaminated by their
urine. Infection in 5–15% of the clinical infections causes
life-threatening manifestations such as acute renal failure
and pulmonary haemorrhage. Fatality among severe cases
is more than 5–40% [4,5]. Leptospirosis is recognized to be
an emerging infectious disease in developed countries due
to outbreaks associated with sporting events [6] and adven-
ture tourism [7–9], and the increasing number of cases found
among travellers [10], participants of recreational activities
[11] and inner-city populations [12]. However, leptospirosis
imparts its greatest disease burden in developing countries
0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2007.05.053
6278
´
E.F. Silva et al. / Vaccine 25 (2007) 6277–6286
[13]. More than 500,000 cases are reported each year [2],
of which the majority occur among rural subsistence farm-
ing populations [1,3,4] and urban slum dwellers [14–16].
Current control measures have been uniformly ineffective in
addressing leptospirosis in these settings [13,16].
Vaccines represent a potentially cost-effective approach to
preventing neglected tropical diseases, such as leptospirosis,
and promoting poverty reduction [17]. An effective lep-
tospirosis vaccine would conceivably prevent human disease
through immunization of at-risk populations or blockade
of transmission through immunization of animal reservoirs.
Leptospirosis is an important veterinary health problem
in domestic cattle, pigs and dogs [1,4,18]. Commercially
available vaccines, consisting of heat or chemically inac-
tivated leptospires, protect hamsters from lethal infection
although protection from sub-lethal infection of the kidneys
is incomplete [19,20]. Yet despite widespread vaccination
with whole-cell inactivated vaccines, leptospirosis remains
prevalent in domestic animal populations [4,21]. Several
problems with current vaccine approaches limit their use in
humans. Whole-cell vaccines produce only short-term immu-
nity, requiring administration semi-annually. Both residual
media components and leptospiral lipopolysaccharride (LPS)
have been associated with adverse reactions [1,3,4]. The vari-
ability of the LPS carbohydrate epitopes accounts for the
serovar specificity of LPS-based vaccines; there is little cross-
protection against infection with the vast majority of other
leptospiral serovars [22–24].
Outer membrane proteins (OMPs) are attractive alter-
natives to whole-cell inactivated vaccines because of
their antigenic conservation across leptospiral species and
serovars. A number of transmembrane and lipoprotein OMPs
have been shown to be surface-exposed and expressed
during infection of the mammalian host [13,25].Inthe
form of purified recombinant proteins, the porin OmpL1
and the lipoproteins LipL41 and LipL32, also known as
hemolysis-associated protein 1, have not been found to
be immunoprotective [26,27]. However, when expressed as
membrane proteins in E. coli, OmpL1 and LipL41 exhibit
synergistic immunoprotection in the hamster model of lep-
tospirosis [27]. Immunization of gerbils with an adenovirus
construct encoding LipL32 improved survival to 87% after
challenge with serovar Canicola compared to 51% survival
in control-immunized gerbils [26]. More recently, immuniza-
tion of gerbils with a pcDNA3.1 DNA vaccine construct
containing the lipL32 gene has also been found to provide
partial protection from lethal challenge [28].
The genes encoding the leptospiral immunoglobulin-like
(Lig) repeat proteins were discovered by screening bacterio-
phage lambda expression libraries with human and equine
leptospirosis sera [29–32]. The Lig proteins belong to a fam-
ily of bacterial immunoglobulin-like (Big) repeat domain
proteins that includes intimin and invasin, the host colo-
nization factors expressed by enteropathogenic E. coli and
Yersinia spp., respectively. Three Lig proteins have been
described, designated LigA, LigB, and LigC. LigA consists
of 13 Ig-like imperfect tandem repeats, while LigB and LigC
have 12 Ig-like tandem repeats followed by large ∼80 kDa
carboxy-terminal domains that do not contain Ig-like repeat
domains. Virulent forms of L. interrogans serovar Copen-
hageni and L. kirschneri serovar Grippotyphosa express LigA
and LigB with sequence-identical amino-terminal regions,
while in both strains the locus encoding LigC is a pseudogene
[30]. A mouse-adapted strain of L. interrogans serovar Mani-
lae expresses LigA and a truncated version of LigB which
includes the tandem Ig-like repeat domains but not the large
carboxy-terminal non-repeat domain [29].
Lig proteins are surface-associated moieties [30] and may
serve as targets for bactericidal responses. Recently, Lig
proteins have been shown to bind fibronectin [33], indi-
cating that they may serve as adhesins. Immunization with
Lig proteins may conceivably induce pathogenesis-blocking
responses. Kozumi et al. demonstrated that immunization
of C3H/HeJ mice, which are genetically deficient of toll-
like receptor 4 [34], with either form of L. interrogans
serovar Manilae-derived LigA protected against lethal chal-
lenge [29]. However, mice are significantly less susceptible
to leptospiral challenge than hamsters, gerbils or guinea pigs,
which are the generally accepted animal models for lep-
tospirosis [4]. More recently, Palaniappan et al. evaluated the
immunoprotective role of recombinant LigA protein in ham-
sters and found that all LigA-immunized animals survived
infection with L. interrogans serovar Pomona [35]. However,
57–88% of the control-immunized animals survived, which
received the same infecting dose (10
8
bacteria) indicating
that the challenge strain was of low virulence. Furthermore,
the study did not have the statistical power to demonstrate
that LigA immunization conferred significantly improved
survival in independent experiments. Therefore there is not
as of yet, sufficient evidence to conclude that recombinant
Lig proteins confer protection in the hamster model.
In this study, we produced recombinant Lig protein frag-
ments and characterized the immune response induced by
immunization with these fragments in hamsters. We found
that a LigA fragment conferred protection against lethal
challenge in an infection model that used a highly viru-
lent L. interrogans strain (LD
50
, 45 bacteria) and showed
that the carboxy-terminal unique region of LigA, corre-
sponding to the last six Ig-like repeat domains, contained
an immunoprotective domain. To our knowledge, this is the
first conclusive evidence demonstrating that immunization
with a purified, recombinant protein confers protection in the
standard Golden Syrian hamster model for leptospirosis.
2. Material and methods
2.1. Leptospira strains and serum samples
L. interrogans serovar Copenhageni strain Fiocruz L1-
130, isolated from a patient during an outbreak of
leptospirosis in the city of Salvador, Brazil [14,36], was culti-
´
E.F. Silva et al. / Vaccine 25 (2007) 6277–6286 6279
vated in Ellinghausen-McCullough-Johnson-Harris (EMJH)
liquid medium (Difco Laboratories) at 29
◦
C. Culture growth
was monitored by counting in a Petroff-Hausser chamber
(Fisher) and dark-field microscopy as described [4]. The clin-
ical isolate was passaged four times in hamsters and three
times in vitro. Seed lots were then prepared and stored in
25% glycerol at −70
◦
C. Frozen aliquots were thawed and
passaged a further four times in EMJH liquid medium prior
to use in experiments to determine the LD
50
in hamsters and
challenge experiments.
Convalescent serum samples were obtained from patients
with laboratory-confirmed leptospirosis during hospital-
based surveillance in Salvador, Brazil [14]. Sera from healthy
control individuals were donated by the state blood bank.
Leptospirosis was confirmed by the microagglutination test
(MAT) as previously described [4,37]. The use of subject sera
for these experiments was approved by the Internal Review
Boards of the Oswaldo Cruz Foundation and New York Pres-
byterian Hospital.
2.2. Cloning, expression and purification of
recombinant 6× His tagged Lig proteins
The proteins LigA and LigB (Fig. 1) were identified
as previously described [30]. The LigANI fragment of
LigA, corresponding to nucleotides 1873–3675 of the ligA
sequence (GenBank accession number NC
005823 Region:
533414–537088), the LigBNI fragment of LigB, correspond-
ing to nucleotides 1873–3773 of the ligB sequence and the
LigBrep fragment of LigB, corresponding to nucleotides
391-1948 of ligB (GenBank accession number NC
005823
Region: 526395–532067), were selected for expression as
recombinant proteins. PCR was used to amplify the target
sequences from genomic DNA purified from L. inter-
rogans Copenhageni Fiocruz L1-130 with the following
primer pairs, LigANI-F 5
-CAATTAAAGATCGTTATACG-
Fig. 1. Schematic representation of LigA and LigB proteins and recombinant
fragments. Boxes represent bacterial immunoglobulin-like (Big) tandem
repeat domains (∼90 amino acids). Amino acids 102–630 (Big domains
2–6 and part of 7) of LigA and LigB, the region with 100% amino acid
sequence identity between these two proteins, are represented as grey boxes.
The C-terminal Big domains of LigA (amino acid position 631–1224) and
LigB (amino acid position 631–1119) have lower amino acid sequence iden-
tity (38%) and are represented as hatched boxes. Lines represent the three
recombinant fragments, LigANI, LigBrep and LigBNI that were cloned and
expressed.
ATAC, LigANI-R 5
-GGTCTAGATTATGGCTCCGTTTT-
AATAGAGG; LigBNI-F 5
-CACCTCCTCTAATACGGA-
TATT, LigBNI-R 5
-TTACACTTGGTTTAAGGAATTAC;
LigBrep-F 5
-ATGGGACTCGAGATTACCGTTACACCA-
GCCATT, LigBrep-R 5
-ATTCCATGGTTATCCTGGA-
GTGAGTGTATTTGT. The resulting 1802 bp (LigANI)
1900 bp (LigBNI) and 1558 bp (LigBrep) PCR products
were cloned into the plasmid pET100-TOPO (Invitrogen)
for expression of Lig recombinant proteins with an N-
terminal 6× His tag. All plasmid constructs were confirmed
by DNA sequencing with an ABI 3100 sequencer (Applied
Biosystems). E. coli BL21(DE3)Star transformants con-
taining the Lig constructs were cultured at 37
◦
C to mid
log phase and expression was induced by isopropyl--d-
thiogalactopyranoside (IPTG), 1 mM final concentration.
The cells were harvested by centrifugation, resuspended in
column buffer (8 M urea, 100 mM Tris, 300 mM NaCl, 5 mM
imidazole, pH 8.0), and disrupted by sonication (3× 1 min
pulses; Sonics & Material Inc.). The soluble fraction was
recovered (10,000 × g, 10 min) and loaded onto Ni
2+
-charged
chelating sepharose columns (Qiagen). Columns contain-
ing bound protein were washed with 10 volumes of column
buffer, then 6× wash buffer (6 M urea, 100 mM Tris, 300 mM
NaCl, pH 8.0) containing 5 mM imidazole for the first three
washes and increasing to 10 mM imidazole for the remain-
ing washes. His-tagged proteins were eluted from the column
with wash buffer containing 250 mM imidazole. An extended
stepwise dialysis procedure was used to remove urea and
imidazole and to promote protein refolding of the recombi-
nant fragments. Dialysis was performed in 18 steps over a
period of 6 days at 4
◦
C with 100 mM Tris, 300 mM NaCl,
pH 8.0 buffer that contained decreasing concentrations of
urea (6–0 M) in each step. After the stepwise procedure, the
purified protein fragment was dialyzed against phosphate-
buffered saline (PBS, pH 7.2) at 4
◦
C for 24 h and stored at
−20
◦
C until use. The Bradford assay (Bio-Rad) was used to
determine protein concentration of purified preparations.
2.3. Protein gel electrophoresis and immunoblotting of
recombinant proteins
For one dimensional sodium dodecyl sulphate-poly-
acrylamide gel electrophoresis (SDS-PAGE), samples were
solublized in sample buffer (62.5 mM Tris hydrochloride
(pH 6.8), 10% glycerol, 5% 2-mercaptoethanol, 2% SDS)
and separated on a discontinuous buffer system (Mini Pro-
tean 3, Bio-Rad). Proteins were transferred to nitrocellulose
membranes following the manufacturer’s instructions (Mini
transblotter, Bio-Rad). Membranes were incubated in block-
ing buffer (0.05 M TBS (pH 7.4), 0.05% (v/v) Tween 20
(TBST), 5% (w/v) non-fat dried milk) overnight at 4
◦
C.
After washing in TBST (3× 5 min per wash), membranes
were incubated with sera (diluted 1:200 in TBST) from lep-
tospirosis patients and healthy control individuals for 1 h
at room temperature. Membranes were washed with TBST
(4× 5 min per wash) and incubated for 1 h at room tempera-
6280
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E.F. Silva et al. / Vaccine 25 (2007) 6277–6286
ture with anti-human IgG conjugated to alkaline phosphatase
(Sigma–Aldrich), which was diluted 1:10,000 in TBST.
Membranes were washed in TBST (3× 5 min per wash) and
TBS before colour development using an NBT/BCIP solution
following the manufacturer’s instructions (Bio-Rad).
2.4. Hamster immunization
Female Golden Syrian hamsters with 4–5 weeks of age
were immunized subcutaneously with Lig recombinant pro-
tein fragments in Freund’s complete adjuvant on day 0 and
a second immunization of antigen in Freund’s incomplete
adjuvant on day 14. Immunization was performed with a
range of recombinant protein doses that included 80/40 g
(first/second immunization); 60/30, 40/20; and 20/10 g.
Emulsions were prepared by mixing protein fragments prepa-
rations in 200–400 l of PBS with an equal volume of
Freunds adjuvant. Hamsters were immunized with a maxi-
mum of 200 l per injection site. A negative control group
of hamsters were immunized with an emulsion of Freund’s
adjuvant and PBS. Pre and post-immunization serum sam-
ples were collected by phlebotomy of the retro-orbital venous
plexus on the day before the first immunization and on the
day before challenge, respectively. All animal studies were
approved by the Committee for the Use of Experimental
Animals of the Oswaldo Cruz Foundation.
2.5. Recombinant Lig ELISA
A preliminary checkerboard analysis was performed to
identify the optimal antigen concentrations and dilutions of
hamster sera and antibody conjugate for the recombinant Lig
protein fragment ELISA. The final protocol was based on the
following conditions. Microtitre plates (Costar) were coated
with 100 ng of recombinant Lig protein in 0.1 M sodium car-
bonate buffer (pH 9.6) at 4
◦
C overnight. The plates were
washed three times with PBS, 0.05% Tween 20 (PBST), and
incubated with 100 l of blocking buffer (PBST, 1% BSA) at
37
◦
C for 1 h. After washing with PBST, wells were incubated
with hamster serum, diluted 1:100 to 1:25,600 in blocking
buffer,at37
◦
C for 1 h. After washing three times with PBST,
wells were incubated with rabbit anti-hamster IgG conju-
gated to horseradish peroxidase (Jackson Immunoresearch
Laboratories), diluted 1:25,000, for 37
◦
C for 1 h. After a
final cycle of washes (two times with PBST and one time
with PBS), 100 lof3,3
,5,5
-tetramethylbenzidine (TMB)
substrate was added to each well. The colour reaction was
allowed to develop for 15 min and stopped with the addi-
tion of 25 lof2MH
2
SO
4
. Absorbance was determined
at 450 nm with a microplate reader (Model 550, Bio-Rad).
Mean values were calculated from serum samples assayed in
duplicate. Each ELISA experiment was repeated three times.
Geometric mean end-point titres (GMT) were determined by
linear regression of the OD
450
values from a serum titration
to obtain a titre at the intersection with the background OD
[38].
2.6. Immunoblotting of native Lig proteins
Late log phase cultures of L. interrogans Fiocruz L1-
130 were incubated in EMJH medium supplemented with
120 mM NaCl to induce LigA and LigB expression as pre-
viously described [39]. Leptospires were harvested, washed
in PBS, resuspended in sample buffer and boiled for 10 min
prior to SDS-PAGE analysis. Membranes of immunoblot-
ted extracts were incubated with sera (diluted 1:200) from
hamsters immunized with recombinant Lig proteins for
1 h at room temperature and after washing were incubated
with Rabbit anti-hamster IgG secondary (diluted 1:5000;
Sigma–Aldrich) for 1 h at room temperature. Membranes
were incubated with goat anti-rabbit IgG conjugate (alka-
line phosphatase; Jackson Immunoresearch Laboratories),
diluted 1:10,000 for 1 h at room temperature and developed
as described in Section 2.3.
2.7. Hamster challenge studies
Challenge experiments were performed with 9-week-old
hamsters in groups of eight to determine the 50% lethal
dose (LD
50
)ofL. interrogans Copenhageni Fiocruz L1-130.
Hamsters were challenged with an inoculum of 10
0
–10
5
lep-
tospires, diluted in PBS and administered intraperitoneally.
Hamsters were monitored three times a day during the 28 day
post-challenge period and euthanized when clinical signs of
terminal disease appeared. The LD
50
was calculated by the
method of Reed–Muench [40].
For vaccine protection experiments, groups of 6–10 ham-
sters, immunized according to protocols described in Section
2, were challenged at age 7–9 weeks with an intraperitoneal
administration of 10
3
leptospires 7 days after the second
immunization. Hamsters were monitored daily for clinical
signs of leptospirosis and euthanized when clinical signs
of terminal disease appeared. Surviving hamsters on day
28 post-challenge were euthanized. Blood, urine, kidney,
lung and liver tissues were harvested for serological, cul-
ture, and histopathology studies. Sterilizing immunity was
determined based on culture isolation of leptospires, identifi-
cation of leptospirosis-associated pathology and histological
detection of leptospires in tissues of surviving hamsters. Kid-
ney tissue and blood samples were used to inoculate EMJH
medium. Dark-field microscopy was performed during an 8
week incubation period to identify positive cultures. Tissue
sections were stained with hematoxylin and eosin for evi-
dence of interstitial nephritis, pulmonary haemorrhage and
liver diffuse dissociation. Warthin–Starry silver staining was
performed to identify leptospires in tissues [41].
A negative control group of hamsters were immunized
with an emulsion of PBS and Freund’s adjuvant and inocu-
lated with a lethal inoculum dose of leptospires according to
the same protocol as described for recombinant Lig protein-
immunized hamsters. A positive control group of hamsters
were immunized with killed whole-leptospires. Washed pel-
lets of cultures L. interrogans strain Fiocruz L1-130 were
´
E.F. Silva et al. / Vaccine 25 (2007) 6277–6286 6281
heat-inactivated 56
◦
C for 20 min, resuspended in PBS and
stored at −20
◦
C until use. Hamsters were immunized with a
dose of 10
9
inactivated leptospires on day 0 and day 14 and
challenged on day 28.
2.8. Statistical analysis
The Student’s t-test was used to determine significant
differences between the geometric mean titres obtained in
ELISA results. The Fisher Exact test and log-rank sum test
were used to determine significant differences for mortal-
ity and survival, respectively, among the groups immunized
with Lig protein fragments and the negative control group.
The chi-squared test for trend was used to evaluate signif-
icant differences between hamster groups immunized with
different doses in the immunoprotection experiments. All P
values were two-sided and a P value of <0.05 was considered
to indicate statistical significance. EpiInfo 6.04d (Centers for
Disease Control and Prevention) and GraphPad Prism 4 soft-
ware systems (GraphPad Software) were used to perform the
statistical analyses.
3. Results
3.1. Preparation of purified recombinant Lig proteins
Three protein fragments were cloned and expressed com-
prising 90% and 60% of the entire LigA and LigB molecules,
respectively (Fig. 1). The LigBrep construct spans the second
to seventh Big repeat domains of the LigA and LigB, a region
of sequence identity between the two proteins. The LigANI
construct spans the 7th to 13th Big repeat domains of the
LigA molecule. The LigBNI construct spans the 7th to 12th
Big repeat domains of the LigB molecule. The sequence iden-
tity between these two non-identical regions is 38%. Cloning
of three PCR products, corresponding to LigANI, LigBNI
and LigBrep, in pET100-TOPO expression vector allowed
for purification of 6–10 mg of His-tagged recombinant pro-
tein per litre of transformed E. coli host culture. However, the
three Lig recombinant fragments were expressed as inclusion
bodies and required denaturing conditions for purification,
followed by prolonged stepwise dialysis to obtain soluble
protein preparations. Protein fragment preparations were
obtained which had over 95% purity (Fig. 2A) and reacted
with pooled leptospirosis patient sera in immunoblot analysis
(Fig. 2B).
3.2. Antibody response induced by immunization with
recombinant Lig protein fragments
Golden Syrian hamsters were immunized with the three
Lig protein fragments in Freund’s adjuvant. Sera obtained
7 days after the second immunization from LigANI and
LigBNI-immunized hamsters demonstrated high titre bind-
ing to homologous recombinant Lig fragments in an ELISA
(Fig. 3A and B). Absorbance values for immune sera were
significantly greater than pre-immune sera at titres up to
1:25,600. Although immunization with LigBrep produced
specific antibodies that recognized the homologous recombi-
nant fragment, absorbance values for immune sera were not
significantly greater than those of pre-immune sera for titres
>1:1600 (Fig. 3C). Absorbance values for serum samples
from hamsters immunized with PBS and Freund’s adjuvant
were <0.06 when diluted 1:100 in ELISAs with the three
recombinant fragments (data not shown). Immunization with
a specific Lig fragment produced significant cross-reactive
antibody responses against the other two fragments. GMTs
ranged from 1:1600 to 1:25,600 in ELISAs that measured
cross-reactive antibodies in sera from immunized hamster to
heterologous Lig proteins (data not shown).
Recombinant protein-based ELISA analysis was per-
formed with sera from hamsters immunized with the LigANI
fragment to determine the duration of humoral immune
response and the response conferred by a range of immu-
nization doses (first/second doses: 20/10, 40/20 and 80/40 g,
Fig. 2. Recombinant Lig proteins and seroreactivity with sera from leptospirosis patients. (A) Coomassie blue-stained SDS-PAGE gel of purified LigANI,
LigBNI and LigBrep recombinant protein fragments (1.5 g/lane). (B) Immunoblot analyses of LigANI, LigBNI and LigBrep recombinant protein fragments.
Membranes were probed with pooled convalescent sera from leptospirosis patients (+) and from healthy individuals (−). Positions of molecular mass markers
(kDa) are shown on the left.
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E.F. Silva et al. / Vaccine 25 (2007) 6277–6286
Fig. 3. Antibody responses in hamsters immunized with recombinant Lig
proteins. ELISA reactions were performed to determine antibody levels
against LigANI (A), LigBNI (B) and LigBrep (C) in sera from hamsters
immunized with LigANI (A: pre-immune, ; immune, ) LigBNI (B: pre-
immune, ; immune, ) and LigBrep (C: pre-immune, ; immune, ).
Mean absorbance (optical density, 450 nm) ±standard deviation (bars) are
shown for a representative experiment among three that evaluated sera from
groups of four immunized hamsters.
Fig. 4). Hamsters produced an antibody response with GMTs
of 1:500–1:1500 one week after primary immunization with
20–80 g of recombinant LigANI. GMTs peaked 1 week
after the second immunization with 10–40 g of recombinant
LigANI (GMT range, 1:8000–1:29,000) and remained ele-
Fig. 4. Kinetics of IgG antibody response in hamsters immunized with
recombinant LigANI protein fragment. ELISA was used to determine anti-
LigANI IgG antibody levels in hamsters immunized on day 0–14 (arrows)
with LigANI doses of 80 and 40 g(), 40 and 20 g(᭹) and 20 and 10 g
(), respectively. Normalized geometric mean endpoint titres (GMTs) and
standard deviation (bars) for sera collected from groups of four hamsters at
weekly intervals over a 63 day follow-up period are shown.
vated (>1:3000) until the last observation point, 63 days after
primary immunization. Of note, the standard deviation was
large (1:4600–1:8700) for sera collected at the 28-day time
point from hamsters immunized with 20/10 g of recom-
binant fragment due to high end-point titres in one animal
(1:18,000). Immunization doses with 80/40 g (first/second
immunization doses) produced significantly higher GMT at
all time points in comparison to 40/20 and 20/10 g immu-
nization schemes.
3.3. Immunization with recombinant Lig proteins
induces antibodies that recognize native Lig proteins
Immunoblot analysis of sera from groups of four hamsters
found that immunization with each of the recombinant Lig
proteins produced antibodies that recognize native Lig pro-
teins in whole Leptospira lysates (Fig. 5). Sera from LigBrep
immunized hamsters reacted with LigA and LigB proteins
while sera from LigANI-immunized hamsters reacted specif-
ically with LigA. Of note, sera from one of four LigBNI
immunized hamsters recognized native LigB, yet all reacted
with native LigA.
3.4. Determination of LD
50
for the L. interrogans
challenge strain and characterization of the hamster
infection model
A series of four challenge experiments found that the mean
LD
50
for L. interrogans serovar Copenhageni strain Fiocruz
L1-130 was 45.9 (±9.3) leptospires in 9-week-old hamsters
(Table 1). Death occurred 10–18 days after challenge and
occurred significantly earlier in groups that received higher
challenge doses. A challenge dose of 10
3
leptospires (∼20×
LD
50
) induced death in all hamsters, which occurred between
´
E.F. Silva et al. / Vaccine 25 (2007) 6277–6286 6283
Fig. 5. Sera from hamsters immunized with recombinant Lig fragments rec-
ognize native LigA and LigB proteins. Immunoblots of whole-cell extracts of
L. interrogans strain Fiocruz L1-130 (10
8
organisms per lane) were probed
with representative samples of pre-immune (PI) and immune sera from ham-
sters immunized with LigANI, LigBNI, LigBrep and PBS (Control) with
Freund’s adjuvant. The mobility of molecular mass markers and expected
positions of LigA (128 kDa) and LigB (201 kDa) are shown on the left and
right side, respectively, of the figure.
the 10th and 18th day post-challenge (Fig. 5). Hamsters devel-
oped the characteristic clinical and histopathologic signs of
leptospirosis. At the time of death, hamsters were jaundiced
and demonstrated bleeding diatheses. Autopsies of infected
hamsters found interstitial nephritis, acute damage of tubu-
lar epithelia, marked dissociation of liver trabecula with
hepatocytes presenting reactive changes such as cytoplas-
mic size variation, prominent nucleoli and binucleation, and
pulmonary haemorrhage as primary pathological findings.
Hamsters are a standard model used to evaluate protective
immunity elicited by whole-cell vaccines [20]. Immunization
with whole Leptospira-based preparations conferred protec-
tion (100%) against lethal challenge (10
3
leptospires) with the
Fiocruz L1-130 strain (Table 2), indicating that the study’s
experimental model reproduced findings reported for estab-
lished models of vaccine-based immunity.
3.5. Protection conferred by immunization with
recombinant Lig protein fragments
Evaluation of recombinant Lig proteins in the hamster
model of vaccine-induced immunity found that immuniza-
tion with the LigANI fragment in Freund’s adjuvant conferred
protection against lethal challenge with 10
3
leptospires (20×
LD
50
)(Fig. 6A and Table 2). Protection conferred with immu-
nization of 80/40 g (first/second immunization dose) of
LigANI ranged from 67% to 100% in the six experiments
in which four different batches of recombinant protein were
used. There were no significant differences between protec-
tion levels between these experiments. Immunization with
Table 1
Determination of the LD
50
for L. interrogans strain Fiocruz L1-130 in the Golden Syrian hamster infection model
Experiment Challenge dose (no. leptospires) LD
50
a
10
5
10
4
10
3
10
2
10
1
10
0
No. deaths per group (n =8)
1 7888ND
b
ND 36.5
2 7886NDND 56.2
3 87853 0 39.8
4 88851 0 51.3
Mean ± S.D.
c
45.9 ± 9.3
a
Fifty percent lethal dose.
b
Not determined.
c
Standard deviation.
Table 2
Protection conferred by immunization with recombinant Lig protein fragments against lethal challenge in the hamster model
Experiment First/second
immunization dose (g)
% Protection (no. survivors/total)
LigANI LigBNI LigBrep Whole-cell preparation
a
Control immunization
b
1 80/40 80.0 (8/10)
c
10.0 (1/10) 0 (0/10) 100 (4/4)
c
− (0/10)
2 80/40 88.9 (8/9)
c
0 (0/9) ND
d
100 (4/4)
c
− (0/9)
3 80/40 66.7 (6/9)
c
22.2 (2/9) ND ND − (0/9)
4 80/40 83.3 (5/6)
c
ND ND ND − (0/6)
5 80/40 70.0 (7/10)
c
ND ND ND − (0/10)
6 80/40 100 (8/8)
c
ND ND ND − (0/8)
60/30 75.0 (6/8)
c
ND ND ND − (0/8)
40/20 87.5 (7/8)
c
ND ND ND − (0/8)
20/10 62.5 (5/8)
c
ND ND ND − (0/8)
a
Immunizations were performed with heat-inactivated preparations of L. interrogans strain Fiocruz L1-130.
b
Control immunizations were performed by administering PBS with Freund’s adjuvant.
c
Protection against lethal challenge was statistically significant (P < 0.05).
d
Not determined.
6284
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E.F. Silva et al. / Vaccine 25 (2007) 6277–6286
the LigBrep and LigBNI fragments did not induce protection
with respect to either mortality or survival.
LigANI-immunized hamsters did not show clinical evi-
dence of infection during the 28 day follow-up period after
challenge infection. Although autopsy examination did not
find macroscopic or histological evidence for disease in Lig-
ANI immunized hamsters that survived lethal challenge and
were sacrificed 28 days post-challenge infection, leptospires
were isolated and/or detected in kidney tissues. In contrast,
leptospires were not isolated and/or detected in tissues of
hamsters immunized with whole Leptospira preparations and
infected with a lethal dose of leptospires, indicating that the
whole-cell vaccine conferred sterile immunity.
Immunization regimens with lower doses (first/second
doses: 20/10, 40/20 and 60/30 g) of LigANI fragment con-
ferred a level of protection (63–88%) against lethal challenge
similar to the initial 80/40 g regimen (Table 2). The level
of vaccine-induced protection was not significantly differ-
ent between groups immunized with different immunization
doses. Among LigANI-immunized hamsters that died after
Fig. 6. Survival of hamsters immunized with recombinant Lig proteins after
lethal challenge. (A) Post-challenge survival is shown for a representative
vaccine evaluation (Experiment 1, Table 2) in which hamsters were immu-
nized on day 21 (80 g) and day 7 (40 g) with LigANI (), LigBNI (),
LigBrep () and PBS () and challenged with L. interrogans strain Fiocruz
L1-130 on day 0. (B) Protective effect conferred by immunization with
increasing LigANI doses (80/40 g, ; 60/30 g, ; 40/20 g, ; 20/10 g,
᭹; and 0/0 g, ) on post-challenge survival (Experiment 6, Table 2).
lethal infection, death occurred significantly later than for the
control group that received a lethal challenge dose (Fig. 6B).
Pre-challenge sera were analyzed from randomly selected
LigANI-immunized hamsters in the six experiments (Table 2)
to determine whether differences in antibody responses cor-
related with outcome after lethal challenge. All pre-challenge
sera from LigANI-immunized hamsters recognized native
LigA protein in immunoblot analysis of whole Leptospira
extracts, regardless of the outcome. In recombinant LigANI-
based ELISA, mean GMTs of pre-challenge sera (1:8000)
from hamsters that died were lower, albeit not significantly
different, than the mean GMTs (1:17,000) of sera from sur-
viving hamsters.
4. Discussion
We have previously shown that Lig proteins are surface-
exposed outer membrane proteins of virulent leptospires
[30] and therefore potential targets of a protective immune
response. Previous studies examined the ability of Lig pro-
teins to confer immunoprotection, yet the conclusions that
could be drawn were limited because of the animal models
and low virulence of the challenge strains used. Kozumi et
al. demonstrated that immunization with recombinant full-
length LigA and truncated LigB proteins protected C3H/HeJ
mice against lethal infection [29]. These findings were the
first to suggest that Lig proteins are immunoprotective moi-
eties. However, the mouse is not an ideal animal model
of leptospirosis because it is relatively resistant to chal-
lenge with pathogenic Leptospira strains and requires a high
inoculum (>10
6
bacteria) to produce lethal infection [42,43].
Palaniappan et al. used hamsters, an accepted model for
leptospirosis, to evaluate LigA protein-induced immunopro-
tection but did not demonstrate a significant difference in
survival between LigA and control-immunized animals in
independent experiments due to low mortality rates (12–43%)
among control-immunized animals [35].
In this study, we evaluated a panel of recombinant Lig
protein fragments in the hamster infection model and found
that the carboxy-terminal region unique to the LigA protein
(LigANI) contained an immunoprotective domain. Fiocruz
L1-130, a highly virulent strain of L. interrogans serovar
Copenhageni was used for which a low inoculum dose
(10
3
bacteria, 20× LD
50
) reproducibly induced characteris-
tic disease manifestations and death in all control-immunized
animals. LigANI conferred an average level of vaccine effi-
cacy (Table 2) which is the highest reported for a subunit
vaccine in the hamster model.
This study provides evidence that a single purified recom-
binant protein is able to induce protective immunity against
lethal challenge in the standard hamster model of leptospiro-
sis. Previous studies using purified recombinant proteins,
including OmpL1, LipL41 and LipL32 [26,27], did not
demonstrate immunoprotection in hamsters, even though
these proteins have been shown to contain immunoprotec-
´
E.F. Silva et al. / Vaccine 25 (2007) 6277–6286 6285
tive epitopes using other approaches [27,28]. Evidence for
LipL32 as a protective antigen comes from studies involving
immunization with recombinant adenovirus [26] and naked
plasmid DNA constructs [28]. The most likely explanation
for the lack of an immunoprotective effect with recombi-
nant proteins is the failure of the proteins to fold correctly
and recapitulate their native secondary structure. Structural
modelling of Lig molecules predicts that the repeat domains
have a highly folded -immunoglobulin sandwich structure
[30]. Lig protein fragments used in this study were puri-
fied under denaturing conditions (8 M urea), and allowed
to renature gradually through a stepwise dialysis method
of urea removal. This approach may have led to the correct
refolding of some but not all of the critical conformational
epitopes present in the Lig molecules, indicating a need for
further refinement of the recombinant protein preparation
protocol.
The mechanism of LigANI vaccine-mediated immunity
remains speculative at this point. Immunization with Lig-
ANI produced robust antibody responses to recombinant and
native protein at the time at which animals were challenged.
Since LigA is a surface-exposed moiety, anti-LigA antibod-
ies may have bactericidal or opsonophagocytic activity. Of
note, the carboxy-terminal unique repeat domain region of
LigA (LigANI) and LigB (LigBNI) has recently been shown
to have fibronectin binding activity [33]. Anti-LigANI anti-
bodies may therefore conceivably block key pathogen–host
cell interactions in disease pathogenesis.
In this study, anti-recombinant LigA antibody titres were
lower in pre-challenge sera from LigANI-immunized ham-
sters that died in comparison to those that survived after
lethal infection, although this difference was not significant.
We interpret this result as an indication that the recom-
binant LigA protein fragment used to immunize hamsters
contains both protective and non-protective epitopes. Eluci-
dation of the role of the anti-LigA antibody response requires
determination whether protective immunity can be passively
transferred. Alternatively, LigA may induce protective cell-
mediated responses as has been observed with whole-cell
vaccines in cattle [44]. Of note, computational analyses found
that LigA contains predicted T-cell epitopes [45].
Whole-cell inactivated leptospiral vaccines were devel-
oped by Ido and colleagues in the early 20th century [46]
and have widespread commercial availability for livestock
and companion animals. These whole-cell vaccines pro-
vide serovar-specific protection against accidental infection
[4,13]. The lack of serovar cross-protection, the need for
annual or biannual revaccination, and a high frequency of side
effects are serious disadvantages that have limited the avail-
ability of whole-cell Leptospira vaccines for human use. A
well-defined subunit vaccine composed of a cross-protective
antigen that results in long-term immunity would be of great
interest in the prevention of human leptospirosis.
Several challenges remain to be addressed to develop
a subunit vaccine based on recombinant Lig proteins. In
this study, immunizations were performed with Freund’s
adjuvant. The efficacy of Lig protein formulations must
be determined using adjuvants acceptable for human use,
such as aluminium hydroxide. An ideal recombinant protein-
based vaccine formulation would comprise a single fragment
that confers cross-protective immunity against infection
with heterologous serovars. Full-length ligA gene sequences
have been identified in a range of L. interrogans and L.
kirschneri serovars [29–32,36]. The predicted amino acid
sequence homology is 80–97% for LigA proteins from these
serovars [30]. Although ligA was not identified in the genome
sequences of L. borgpetersenii serovar Hardjo [47] and L.
interrogans serovar Lai [48], individual Big domains in LigA
and LigB proteins share an amino acid sequence identity
of 17–99% with each other. In this study, immunization
with the LigANI fragment produced cross-reactive antibody
responses against LigBrep and LigBNI fragments, which
in turn may have contributed to the protective immunity
observed in the hamster trials. In summary, we have shown
that a recombinant protein fragment of the putative viru-
lence determinant, LigA, reproducibly conferred high-level
protection against lethal infection in the hamster model for
leptospirosis. These findings provide proof-of-concept evi-
dence for the feasibility of developing a subunit vaccine for
human leptospirosis.
Acknowledgements
This work was supported by the Brazilian National
Research Council (grants 300861/1996, 01.06.0298.00 and
420067/2005, and 554788/2006); Bio-Manguinhos, Oswaldo
Cruz Foundation, Brazilian Ministry of Health (grant
09224-7); VA Medical Research Funds; the National Insti-
tute of Allergy and Infectious Diseases (grants AI052473
and AI034431) and Fogarty International Centre (grant
TW00919).
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